Modular point-of-care devices, systems, and uses thereof

ABSTRACT

The present invention provides devices and systems for use at the point of care. The methods devices of the invention are directed toward automatic detection of analytes in a bodily fluid. The components of the device are modular to allow for flexibility and robustness of use with the disclosed methods for a variety of medical applications.

This application is a continuation application of, and claims priorityunder 35 U.S.C.§120 to, U.S. patent application Ser. No. 13/889,674filed May 8, 2013, which is a continuation of U.S. patent applicationSer. No. 13/326,023 filed Dec. 14, 2011, which is a continuation of U.S.patent application Ser. No. 12/244,723, filed Oct. 2, 2008, now U.S.Pat. No. 8,088,593, which is a non-provisional application claimingpriority under 35 U.S.C. §119(e) to, and which claims the benefit ofU.S. Provisional Application No. 60/997,460, filed Oct. 2, 2007, thecontents of all of which non-provisional and provisional applicationsare hereby incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

The discovery of a vast number of disease biomarkers and theestablishment of miniaturized medical systems have opened up new avenuesfor the prediction, diagnosis and monitoring of treatment of diseases ina point-of-care setting. Point-of-care systems can rapidly deliver testresults to medical personnel, other medical professionals and patients.Early diagnosis of a disease or disease progression can allow medicalpersonnel to begin or modify therapy in a timely manner.

Multiplexed biomarker measurement can provide additional knowledge ofthe condition of a patient. For example, when monitoring the effects ofa drug, three or more biomarkers can be measured in parallel. Typically,microtiter plates and other similar apparatuses have been used toperform multiplexed separation-based assays. A microtiter plate (forexample, a 384 well microtiter plate) can perform a large number ofassays in parallel.

In a Point-of-Care (POC) device, the number of assays that can beperformed in parallel is often limited by the size of the device and thevolume of the sample to be analyzed. In many POC devices, the numberassays performed is about 2 to 10. A POC device capable of performingmultiplexed assays on a small sample would be desirable.

A shortcoming of many multiplexed POC assay devices is the high cost ofmanufacturing the components of the device. If the device is disposable,the high cost of the components can make the manufacturing of a POCdevice impractical. Further, for multiplexed POC devices thatincorporate all of the necessary reagents onboard of the device, if anyone of those reagents exhibit instability, an entire manufactured lot ofdevices may have to be discarded even if all the other reagents arestill usable.

When a customer is interested in a customizing a POC device to aparticular set of analytes, manufacturers of multiplexed POC assaysystems are often confronted with a need to mix-and-match the assays andreagents of the device. A multiplexed POC assay suitable to eachcustomer can be very expensive, difficult to calibrate, and difficult tomaintain quality control.

POC methods have proven to be very valuable in monitoring disease andtherapy (for example, blood glucose systems in diabetes therapy,Prothrombin Time measurement in anticoagulant therapy using Warfarin).By measuring multiple markers, it is believed that complex diseases(such as cancer) and therapies such as multi-drug therapy for cancer canbe better monitored and controlled.

SUMMARY OF THE INVENTION

Thus, there remains an unmet need for alternative designs of POCdevices. A desirable design provides modular capture surfaces and assayincubation elements. Furthermore, modular capture surfaces and assayincubation elements need to be integrated into POC disposables suitedfor just-in-time (JIT) manufacturing methods. It would be desirable toprovide a customizable POC device at a practical cost to user and themanufacturer. The present invention addresses these needs and providesrelated advantages as well.

In an aspect, a cartridge is disclosed for automated detection of ananalyte in a bodily fluid sample comprising: an array of addressableassay units configured to run a chemical reaction that yields adetectable signal indicative of the presence or absence of the analyte;and an array of addressable reagent units, wherein an individualaddressable reagent unit of the array is addressed to correspond to anindividual addressable assay unit of the array of assay units, andwherein the individual reagent units are configured to be calibrated inreference to the corresponding individual assay unit before the arraysare assembled on the cartridge. The device can further comprise a samplecollection unit configured to receive the bodily fluid sample.

In another aspect, a cartridge is disclosed for automated detection ofan analyte in a bodily fluid sample comprising: a sample collection unitconfigured to receive the bodily fluid sample; an array of assay unitsconfigured to receive a portion of the sample from the sample collectionunit and run a chemical reaction that yields a detectable signalindicative of the presence of the analyte in the sample; and an array ofreagent units containing reagents for running the chemical reaction;wherein an individual assay unit of the array of assay units and anindividual reagent unit of the array of reagents units are configured tobe movable into fluid communication such that reagents for running thechemical reaction are brought to contact with the bodily fluid sample inthe assay unit.

An individual reagent unit can be configured to receive a movable assayunit. In some embodiments, the individual assay unit comprises an assaytip. In some embodiments, the individual assay unit is configured to runan immunoassay.

The bodily fluid sample can be a blood sample. In some instances, asample collection unit is configured to receive a volume of the bodilyfluid sample about 50, 20, 10, 5 or 3 microliters or less. In aninstance, the sample collection unit is configured to receive a volumeof the bodily fluid sample equivalent to a single drop of blood.

A device as described herein can comprise a pretreatment unit configuredto retrieve a portion of the bodily fluid sample for running thechemical reaction to detect the analyte and the pretreatment unit can beconfigured to retrieve plasma from whole blood sample received in thesample collection unit.

In an aspect, a system is described herein for automated detection of ananalyte in a bodily fluid sample comprising: a device as describedherein; and a detection assembly for detecting the detectable signalindicative of the presence or absence of the analyte. The system canfurther comprise a programmable mechanical device configured to move theindividual assay unit from a first location to a second location. Insome instances, a system comprises a fluid transfer device. The fluidtransfer device can be a pipette and can be automated. A system can alsocomprise a communication assembly for transmitting a protocol based onthe analyte to be detected. In some instances, a system herein comprisesa heating block configured to receive an individual assay unit and canalso comprise a magnetic block, for example, that can be used forseparation of red cells from the sample.

In another aspect, a system is disclosed for automated detection of aplurality of analytes in a bodily fluid sample, comprising: a fluidicdevice comprising: a sample collection unit configured to contain thebodily fluid sample; an array of assay units, wherein an individualassay unit of said array of assay units is configured to run a chemicalreaction that yields a signal indicative of an individual analyte ofsaid plurality of analytes being detected; and an array of reagentunits, wherein an individual reagent unit of said array of reagent unitscontains a reagent; and a fluid transfer device comprising a pluralityof heads, wherein an individual head of the plurality of heads isconfigured to engage the individual assay unit, and wherein said fluidtransfer device comprises a programmable processor configured to directfluid transfer of the bodily fluid sample from the sample collectionunit and the reagent from the individual reagent unit into theindividual assay unit. In some embodiments, the configuration of theprocessor to direct fluid transfer effects a degree of dilution of thebodily fluid sample in the array of assay units to bring signalsindicative of the plurality of analytes being detected within adetectable range, such that said plurality of analytes are detectablewith said system.

In some instances, a bodily fluid sample comprises at least two analytesthat are present at concentrations that differ by at least 2, 5, 10, 15,50, or 100 orders of magnitude. The degree of dilution of the bodilyfluid sample can bring the signals indicative of the at least twoanalytes within the detectable range.

A system herein can further comprise a detector configured to detectsignal intensities of the detectable range. An exemplary detector is aphotomultiplier and a detectable range of the detector can be about 20to about 10 million counts.

In some embodiments, wherein the individual head of a fluid transferdevice is configured to adhere to the individual assay unit. Theindividual assay unit can provide an immunoassay reaction site. In someinstances, the individual assay unit is a pipette tip. The fluidtransfer device can be a pipette such as an air-displacement pipette.The fluid transfer device can also comprises a motor in communicationwith the programmable processor, wherein the motor can move saidplurality of heads based on a protocol from said programmable processor.

In another aspect, a system is described herein for automated detectionof a plurality of analytes in a plasma portion of a whole blood sample,comprising: a device configured to automatically receive and process thewhole blood sample to yield the plasma portion, from which a detectablesignal indicative of the presence or absence of the analyte of interestis generated onboard the device; and a detection assembly for detectingthe detectable signal indicative of the presence or absence of theanalyte.

In an aspect, provided herein is a method of detecting an analyte in abodily fluid sample comprising: providing a blood sample to a device asdescribed herein; allowing said sample to react within at least oneassay unit; and detecting said detectable signal generated from saidanalyte collected in said sample of bodily fluid. The bodily fluidsample can be blood and the method can comprise retrieving plasma fromthe blood.

In an aspect as provided herein, a method of on-demand assembly of acartridge for automated detection of an analyte in a bodily fluidsample, wherein the device comprises a housing, said housing comprising:an array of addressable assay units, wherein an individual assay unit ofthe array is configured to run a chemical reaction that yields adetectable signal indicative of the presence or absence of the analyte;and an array of addressable reagent units, wherein an individual reagentunit of the array is addressed to correspond to the individual assayunit, said method comprises: (i) placing according to the analyte to bedetected an array of addressable assay units, wherein an individualassay unit of the array is configured to run a chemical reaction thatdetects an analyte of interest ordered by said end user, into thehousing; (ii) placing according to the analyte to be detected an arrayof reagent units, wherein an individual reagent unit of the arraycorresponds to the individual assay unit, into the housing; and (iii)securing the arrays of (i) and (ii) within the housing of the device.The method can comprise selecting an analyte to be detected. In someembodiments, the method comprises sealing the cartridge. In anembodiment, the method comprises labeling the cartridge with a readablelabel indicating the analyte to be detected, for example with a bar codeor RFID.

In an aspect, a method is provided for automated detection of aplurality of analytes in a bodily fluid sample, comprising: providingthe bodily fluid sample to a fluidic device, wherein the fluidic devicecomprises: a sample collection unit configured to contain the bodilyfluid sample; an array of assay units, wherein an individual assay unitof said array of assay units is configured to run a chemical reactionthat yields a signal indicative of an individual analyte of saidplurality of analytes being detected; and an array of reagent units,wherein an individual reagent unit of said array of reagent unitscontains a reagent; engaging the individual assay unit using a fluidtransfer device; transferring the bodily fluid sample from the samplecollection unit to the individual assay unit using the fluid transferdevice; and transferring the reagent from the individual reagent unit tothe individual assay unit, thereby reacting the reagent with the bodilyfluid sample to yield the signal indicative of the individual analyte ofthe plurality of analytes being detected.

In an embodiment, the fluid transfer device comprises a plurality ofheads, wherein an individual head of the plurality of heads isconfigured to engage the individual assay unit; and wherein said fluidtransfer device comprises a programmable processor configured to directfluid transfer of the bodily fluid sample from the sample collectionunit and the reagent from the individual reagent unit into theindividual assay unit. The method can further comprise providinginstructions to the programmable processor, wherein the instructions candirect the step of transferring the bodily fluid sample to theindividual assay unit.

In an embodiment, the step of transferring the bodily fluid sampleeffects a degree of dilution of the bodily fluid sample in theindividual assay unit to bring the signal indicative the individualanalyte of the plurality of analytes being detected within a detectablerange. The bodily fluid sample can comprise at least two individualanalytes that are present at concentrations that differ by at least 2,5, 10, 15, 50, or 100 orders of magnitude. In some instances, the degreeof dilution of the bodily fluid sample brings the signals indicative ofthe at least two individual analytes within the detectable range. In anembodiment, the detectable range is about 1000 to about 1 million countsper second using a photomultiplier.

In an embodiment, the reagent in the individual reagent unit is anenzyme substrate for an immunoassay and the method can further compriserepeating the step of transferring the reagent from the individualreagent unit after the reaction to yield the signal indicative of theindividual analyte of the plurality of analytes being detected iscomplete, thereby creating a second reaction to yield a second signalindicative of the individual analyte. An intensity of the signal and asecond intensity of the second signal indicative of the individualanalyte can be averaged to calculate the final intensity of the signalindicative of the individual analyte.

In an aspect, a method is described herein of measuring a volume of aliquid sample, comprising: reacting a known quantity of a controlanalyte in a liquid sample with a reagent to yield a detectable signalindicative of the control analyte; and comparing said detectable signalwith an expected detectable signal, wherein the expected signal isindicative of an expected volume of the liquid sample, and wherein saidcomparison provides a measurement of said volume of said liquid samplebeing measured. In some instances, the control analyte is not normallypresent in said liquid sample in a detectable amount. The method cancomprise verifying the volume of said liquid sample when the measurementof the volume of the sample is within about 50% of the expect volume ofthe liquid sample. In an embodiment, the method further comprises:reacting a bodily fluid sample containing a target analyte with areagent to yield a detectable signal indicative of the target analyte;and measuring the quantity of the target analyte in the bodily fluidsample using an intensity of said detectable signal indicative of thetarget analyte and the measurement of said volume of said liquid sample.The liquid sample and the bodily fluid sample can be the same sample andthe control analyte does not react with the target analyte in the bodilyfluid sample. In some instances, the liquid sample and the bodily fluidsample are different liquid samples. The control analyte can be, forexample, fluorescein-labeled albumin, fluorescein labeled IgG,anti-fluorescein, anti-digoxigenin, digoxigenin-labeled albumin,digoxigenin-labeled IgG, biotinylated proteins, non-human IgG.

In another aspect, a method of retrieving plasma from a blood sample isprovided herein that comprises: mixing a blood sample in the presence ofmagnetizable particles in a sample collection unit, wherein themagnetizable particles comprise an antibody capture surface for bindingto non-plasma portions of the blood sample; and applying a magneticfield above a plasma collection area to the mixed blood sample to effectsuspension of the non-plasma portions of the blood sample on top of theplasma collection area. In some instances, the sample collection unit isa capillary tube. The blood sample can be less than about 20 microlitersand the plasma retrieved can be less than about 10 microliters. In someinstances, the blood sample is not diluted. In some instance, mixingoccurs in the presence of antibodies unbound to a solid surface. Themixing can comprise mixing by syringe action.

In yet another aspect, a method is provided herein of using automatedimmunoassay for detecting an analyte present in plasma portion of awhole blood sample, comprising: providing a whole blood sample to adevice that is configured to automatically receive and process on boardthe whole blood sample to yield the plasma portion, from which adetectable signal indicative of the presence or absence of the analyteof interest is generated on board; detecting said signal that isindicative of the presence or absence of the analyte in said bodilyfluid sample; and transmitting result of (b) to an end user. Theimmunoassay can be an ELISA. In some instances, the result istransmitted wirelessly.

In some embodiments, a method as described herein is carried out in asystem as described herein.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Many novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which many principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates an exemplary device of the invention comprising assayunits, reagents unit, and other modular components of the device.

FIG. 2 illustrates two side-cut away views of the exemplary device ofFIG. 1 comprising cavities in the housing of the device shaped toaccommodate an assay unit, a reagent unit, and a sample tip.

FIG. 3A demonstrates an exemplary assay unit that comprises a small tipor tubular formation.

FIG. 3B demonstrates an example of a sample tip as described herein.

FIGS. 4A and 4B illustrate two examples of a reagent unit comprising acup.

FIG. 5 demonstrates an example of a system comprising a device and afluid transfer device.

FIG. 6 illustrates an exemplary system of the invention comprising aheating block for temperature control and a detector.

FIG. 7 demonstrates an exemplary a system wherein a patient deliversblood to a device and then the device is inserted into a reader.

FIG. 8 illustrates the process flow of building a system for assessingthe medical condition of a patient.

FIGS. 9A through 9E demonstrate an example of a plasma separation methodwherein a whole blood sample has been aspirated into a sample tip and amagnetic reagent is mixed and suspended with the sample, then a magneticfield is applied to the whole blood sample and magnetic reagent mixture.Separated blood plasma sample can then be distributed into a well of adevice.

FIG. 10 demonstrates an exemplary method of a control assay as describedherein comprising a known quantity of control analyte.

FIG. 11 illustrates a thin film, for example, contamination, within thetip when a liquid is expelled and another liquid aspirated.

FIG. 12 illustrates a calibration curve correlating an assay unit and areagent unit for conducting an assay for VEGFR2.

FIG. 13 illustrates a calibration curve correlating results for an assayunit and a reagent unit for conducting an assay for P1GF in a system, asmeasured with a luminometer.

FIG. 14 illustrates CRP concentration plotted against the assay signal(photon counts) and the data fitted to a 5-term polynomial function togenerate a calibration function.

FIG. 15 shows a fit was achieved between a model and the values of theparameters Smax, C0.5 and D as described herein.

FIG. 16 displays data according to the dilution used to achieve thefinal concentration in an assay tip.

FIG. 17 illustrates the normalized assay response (B/Bmax) is plottedagainst the log normalized concentration (C/C0.5) for relativedilutions: 1:1 (solid line), 5:1 (dashed line), and 25:1 (dotted line).

FIGS. 18 and 19 illustrate a similar example as FIG. 17 at differentnormalized concentrations.

FIG. 20 demonstrates the assay response for a control analyte after thesteps of: removal of the detector antibody, washing the assay, andadding a substrate, as read in a spectro-luminometer for 0.5 s.

FIG. 21 demonstrates the results of an assay that was evaluated bymeasuring photons produced over about 10 s in a system herein.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments and aspects of the invention described herein pertain todevices, systems, and methods for automated detection of an analyte in asample of bodily fluid. The invention is capable of detecting and/orquantifying analytes that are associated with specific biologicalprocesses, physiological conditions, disorders or stages of disorders,or effects of biological or therapeutic agents. The embodiments andexamples of the invention described herein are not intended to limit thescope of invention.

Devices

In an aspect of the invention, a device for automated detection of ananalyte in a bodily fluid sample comprises an array of addressable assayunits configured to run a chemical reaction that yields a detectablesignal indicative of the presence or absence of the analyte, and anarray of addressable reagent units, each of which is addressed tocorrespond to one or more addressable assay units in said device, suchthat individual reagent units can be calibrated in reference to thecorresponding assay unit(s) before the arrays are assembled on thedevice.

In another aspect of the invention, a device for automated detection ofan analyte in a bodily fluid sample comprises an array of assay unitsconfigured to run a chemical reaction that yields a detectable signalindicative of the presence of the analyte, and an array of reagent unitscontaining reagents for running the chemical reaction, wherein at leastone of the assay units and at least one of the reagent units are movablerelative to each other within the device such that reagents for runningthe chemical reaction are automatically brought to contact with thebodily fluid sample in the assay unit.

In an embodiment of a device of the invention, the array of assay unitsor reagent units can be addressed according to the chemical reaction tobe run by the configured assay unit. In another embodiment, at least oneof the assay units and at least one of the reagent units are movablerelative to each other within the device such that reagents for runningthe chemical reaction are automatically brought to contact with thebodily fluid sample in the assay unit.

In one embodiment, the device of the invention is self-contained andcomprises all reagents, liquid- and solid-phase reagents, required toperform a plurality of assays in parallel. Where desired, the device isconfigured to perform at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 100, 200, 500, 1000 or more assays. One or more control assays canalso be incorporated into the device to be performed in parallel ifdesired.

The assays can be quantitative immunoassays and can be conducted in ashort period of time. Other assay type can be performed with a device ofthe invention including, but not limited to, measurements of nucleicacid sequences and measurements of metabolytes, such as cholesterol. Insome embodiments, the assay is completed in no more than one hour,preferably less than 30, 15, 10, or 5 minutes. In other embodiments, theassay is performed in less than 5 minutes. The duration of assaydetection can be adjusted accordingly to the type of assay that is to becarried out with a device of the invention. For example, if needed forhigher sensitivity, an assay can be incubated for more than one hour orup to more than one day. In some examples, assays that require a longduration may be more practical in other POC applications, such as homeuse, than in a clinical POC setting.

Any bodily fluids suspected to contain an analyte of interest can beused in conjunction with the system or devices of the invention.Commonly employed bodily fluids include but are not limited to blood,serum, saliva, urine, gastric and digestive fluid, tears, stool, semen,vaginal fluid, interstitial fluids derived from tumorous tissue, andcerebrospinal fluid.

A bodily fluid may be drawn from a patient and provided to a device in avariety of ways, including but not limited to, lancing, injection, orpipetting. As used herein, the terms subject and patient are usedinterchangeably herein, and refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets. In oneembodiment, a lancet punctures the skin and withdraws a sample using,for example, gravity, capillary action, aspiration, or vacuum force. Thelancet may be part of the device, or part of a system, or a stand alonecomponent. Where needed, the lancet may be activated by a variety ofmechanical, electrical, electromechanical, or any other known activationmechanism or any combination of such methods. In another embodimentwhere no active mechanism is required, a patient can simply provide abodily fluid to the device, as for example, could occur with a salivasample. The collected fluid can be placed in the sample collection unitwithin the device. In yet another embodiment, the device comprises atleast one microneedle which punctures the skin. 3

The volume of bodily fluid to be used with a device is generally lessthan about 500 microliters, typically between about 1 to 100microliters. Where desired, a sample of 1 to 50 microliters, 1 to 40microliters, 1 to 30 microliters, 1 to 10 microliters or even 1 to 3microliters can be used for detecting an analyte using the device.

In an embodiment, the volume of bodily fluid used for detecting ananalyte utilizing the subject devices or systems is one drop of fluid.For example, one drop of blood from a pricked finger can provide thesample of bodily fluid to be analyzed with a device, system or methoddescribed herein.

A sample of bodily fluid can be collected from a subject and deliveredto a device of the invention as described hereinafter.

In an embodiment, the arrays of assay and reagent units are configuredto be a set of mix-and-match components. The assay units can comprise atleast one capture surface capable of reacting with an analyte from thesample of bodily fluid. The assay unit may be a tubular tip with acapture surface within the tip. Examples of tips of the invention aredescribed herein. A reagent unit typically stores liquid or solidreagents necessary for conducting an assay that detect a give analyte.Each individual assay and reagent unit can be configured for assayfunction independently. To assemble a device, the units can be assembledin a just-in-time fashion for use in integrated cartridges.

Separate components, both liquid and solid phase, can be made and thenbe tested for performance and stored. In an embodiment, the assembly ofthe device is carried out in on-demand fashion at a manufacturinglocation. The device can be modular and include components such as ahousing that is generic for all assays, assay units, such as tips, andreagent units, such as a variety of frangible or instrument operablecontainers that encapsulate liquid reagents. In some instances, anassembled device is then tested to verify calibration (the relation ofthe system response to known analyte levels). Assay devices can beassembled from a library of pre-manufactured and calibrated elements ondemand. In some embodiments, fluidic pathways within a device can besimple and obviate any chance of trapping bubbles and providing anefficient way to wash away excess labeled reagents in reagent excessassays such as ELISAs.

A housing for a device of the invention can be made of polystyrene oranother moldable or machinable plastic and can have defined locations toplace assay units and reagent units. In an embodiment, the housing hasmeans for blotting tips or assay units to remove excess liquid. Themeans for blotting can be a porous membrane, such as cellulose acetate,or a piece bibulous material such as filter paper.

In some embodiments, at least one of the components of the device may beconstructed of polymeric materials. Non-limiting examples of polymericmaterials include polystyrene, polycarbonate, polypropylene,polydimethysiloxanes (PDMS), polyurethane, polyvinylchloride (PVC),polysulfone, polymethylmethacrylate (PMMA),acrylonitrile-butadiene-styrene (ABS), and glass.

The device or the subcomponents of the device may be manufactured byvariety of methods including, without limitation, stamping, injectionmolding, embossing, casting, blow molding, machining, welding,ultrasonic welding, and thermal bonding. In an embodiment, a device inmanufactured by injection molding, thermal bonding, and ultrasonicwelding. The subcomponents of the device can be affixed to each other bythermal bonding, ultrasonic welding, friction fitting (press fitting),adhesives or, in the case of certain substrates, for example, glass, orsemi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components.

An exemplary device as described herein is illustrated in FIG. 1. Thedevice 100 is also sometimes referred to herein as a cartridge 100. Thedevice 100 comprises a housing 130 with locations to accommodate assayunits 121 and reagent units 103, 122, 124, 125. In the exemplaryembodiment of FIG. 1, assay units 121 occupy a center row of the housing130 of the device 100. The assay units 121 can optionally include atleast one calibration unit 126. In an example, the assay units 121 aresimilar to pipette tips and are referred to as assay tips 121 and thecalibration units 126 are referred to as calibration tips 126 herein,however, the assay units 121 can be of any shape and size as areaccommodated broadly by a device 100 as described herein. The assayunits 121 and calibration units 126 are exemplary assay units 121 andare described in more detail herein. The assay units 121 in FIG. 1 cancomprise a capture surface and are capable, for example, of performing achemical reaction such as nucleic acid assays and immunoassays. Theassay units 121 can be assembled into the housing according toinstructions or the assays that a user wishes to perform on a sample.

As shown in FIG. 1, the housing of the device 100 can comprise a samplecollection unit 110 configured to contain a sample. A sample, such as ablood sample, can be placed into the sample collection unit 110. Asample tip 111 (for example, a pipette tip that couples to a fluidtransfer device as described in more detail herein) can occupy anotherportion of the housing 130. When an assay is to be run the sample tip111 can distribute the sample to pretreatment reagent units orpretreatment units 103, 104, 105, 106, 107, or assay units 121.Exemplary pretreatment units 103, 104, 105, 106, 107 include but are notlimited to: mixing units 107, diluent or dilution units 103, 104, and,if the sample is a blood sample, plasma removal or retrieval units 105,106. The pretreatment units 103, 104, 105, 106, 107 can be the same typeof unit or different types of units. Other pretreatment units 103, 104,105, 106, 107 as are necessary to run a chemical reaction can beincorporated into device 100 as would be obvious to one skilled in theart with knowledge of this disclosure. The units 103, 104, 105, 106, 107can contain various amounts of reagents or diluents, flexible towhatever is needed to run the assay on the current cartridge 100.

Often, the assay units 121 can be manufactured separately from thehousing 130 and then inserted into the housing 130 with pick-and-placemethods. The assay units 121 can fit snugly into the housing 130 or canfit loosely into the housing 130. In some embodiments, the housing 130is manufactured such that is holds the reagent units 103, 122, 124, 125and/or assay units 121 snugly in place, for example during shipping ormanipulation a cartridge. Reagents units 103, 122, 124, 125 are shown inFIG. 1 that contain a conjugate reagent 122 (for example, for use withan immunoassay), a wash reagent 125 (for example, to wash said conjugatefrom capture surfaces), and a substrate 124 (for example, an enzymesubstrate). Other embodiments of the device 100 and the components inthe example in FIG. 1 are described herein. Reagent units 103, 122, 124,125 can be manufactured and filled separately from the housing 130 andthen placed into the housing 130. In this way, a cartridge 100 can bebuilt in a modular manner, therefore increasing the flexibility of thecartridge 100 to be used for a variety of assays. Reagents in a reagentunit 103, 122, 124, 125 can be chosen according to the assay to be run.Exemplary reagents and assays are described herein.

A device, such as the example shown in FIG. 1, can also comprise otherfeatures as may be needed to run a chemical reaction. For example, ifthe assay units 121 are assay tips 121 as described herein, the devicecan comprise tip touch-off pads 112 to remove excess sample or reagentfrom an assay tip 121 or a sample tip 111 after fluid transfer, forexample, by a system as described herein. The housing 130 can alsocomprise units or areas 101, 102 within the device 100 for placing aused tip or unit, for example, in order to avoid cross-contamination ofa sample tip 111 or assay unit 121. In FIG. 1, the device 100 comprisesa sample tip 111 for transferring a sample between units of the device100. The device 100 as illustrated in FIG. 1 also comprises apretreatment tip 113 for transferring a sample that has been pretreatedin a unit of the device 100 to other units of a device 100 to perform achemical reaction. For example, the sample tip 111 can be used to removea blood sample from the sample collection unit 110 and transfer theblood sample to pretreatment units 103, 104, 105, 106, 107 as described.Red cells can be removed from the blood sample in the pretreatment units103, 104, 105, 106, 107 and the pretreatment tip 113 can then be used tocollect the blood plasma from the pretreatment units 103, 104, 105, 106,107 and transfer the blood plasma to another pretreatment unit (forexample, a diluent unit) 103, 104, 105, 106, 107 and/or to at least oneassay unit 121. In an embodiment, a sample tip 111 is the samplecollection unit 110. In another embodiment, the sample collection unit110 is similar to a well and is configured to contain a sample asreceived by a user.

Assay units 121 and reagent units 103, 122, 124, 125 as shown in FIG. 1can be addressable to indicate the location of the units on thecartridge 100. For example, a column of the cartridge 100 as shown inFIG. 1 can contain an assay unit 121 to run an assay configured todetect C-reactive protein, and the column can contain correspondingreagent units 103, 122, 124, 125 for that assay in the same column,wherein the units are addressed to correspond to each other. Forexample, the addresses can be entered and stored in a computer system,and the cartridge 100 can be given a label, such as a bar code. When thebar code of the cartridge 100 is scanned for use, the computer systemcan send the addresses of the units to a system, such as those describedherein, to transfer the fluids and run a reaction according to theaddresses entered into the computer. The addresses can be part of aprotocol sent to operate the system. The addresses can be in anyconfiguration and can be altered if need be to change the protocol ofrunning an assay, which in turn can offer a change in assay protocol orsteps to a user of the cartridge that has not been typically availablein prior art POC devices. In some embodiments, the housing 130 and unitsare configured in a 6 by 8 array of units as shown in FIG. 1. The layoutof the units can be of any format, for example, rectangular arrays orrandom layouts. A cartridge 100 can comprise any number of units, forexample between 1 and about 500. In some embodiments, a cartridge 100has between 5-100 units. As an example as shown in FIG. 1, the cartridge100 has 48 units.

Two side cut-away views of the exemplary device 200 of FIG. 1 areillustrated in FIGS. 2A and 2B. A cavity can be shaped in a housing 220of a device to accommodate assay units (for example, assay tips) 201 ina vertical orientation (housing horizontal) with their bosses toward thetop of the device 200. As shown in FIG. 2, a cavity can also be shapedto accommodate a reagent unit 210, 212 or a sample collection unit ortip 202. There may be features in the housing 220 to capture the unitsprecisely and hold them securely. Such features can also be designed tooperate with a mechanism for moving the tips, such as tip pick-up anddrop-off. In another embodiment, the sample collection unit comprises abendable or breakable element that serves to protect a small collectiontube during shipment and to hold a plunger device in place within acapillary. Also shown in FIG. 2A are two exemplary embodiments ofreagent units 210, 212 as are described herein. The bottom of thehousing 220 can be configured to collect waste liquids, for example,wash reagents after use that are transferred back through a hole in thehousing 220 to the bottom. The housing 220 can comprise an absorbent padto collect waste fluids. The assay units 201 and sample units 202 can bepositioned to fit through a cavity of the housing 220 of the device 200and extend beyond an inner support structure. The reagent units 210, 212fit snugly into the housing as is shown in FIG. 2 and do not extendbeyond the inner support structure. The housing 220 and the areas inwhich the assay units 201 and reagents units 210, 212 can be held andpositioned may adapt a variety of patterns.

In some embodiments, each tip provides for a single assay and can bepaired with or corresponded to an appropriate reagent, such as requiredreagents for running the designated assay. Some tips provide for controlassay units and have known amounts of analyte bound to their capturesurfaces either in the manufacturing process or during the performanceof an assay. In case of a control assay unit, the unit is configured torun a control assay for comparison. The control assay unit may comprise,for example, a capture surface and analyte that are in a solid or liquidstate.

In many embodiments, the device holds all reagents and liquids requiredby the assay. For example, for a luminogenic ELISA assay the reagentswithin the device may include a sample diluent, a detector conjugate(for example, three enzyme-labeled antibodies), a wash solution, and anenzyme substrate. Additional reagents can be provided as needed.

In some embodiments, reagents can be incorporated into a device toprovide for sample pretreatment. Examples of pretreatment reagentsinclude, without limitation, white cell lysis reagents, reagents forliberating analytes from binding factors in the sample, enzymes, anddetergents. The pretreatment reagents can also be added to a diluentcontained within the device.

An individual reagent unit can be configured to receive a movable assayunit. In some embodiments, the individual assay unit comprises an openended hollow cylindrical element comprising a capture surface and areaction cuvette. A cylindrical assay unit can be referred to as anassay tip herein. In some embodiments, the individual assay unit isconfigured to run an immunoassay. An assay unit 301 that comprises asmall tip or tubular formation is shown in FIG. 3A. In some instances,the tip 301 is configured to provide an interior cylindrical capturesurface 311 and a boss 321 capable of engaging with the housing ofdevice. In some instances, the boss 321 and the tip 301 is configured toengage with a mechanism of moving the tip 301 such as a system asdescribed herein or for example, a fluid transfer device. An assay tip301 as shown in FIG. 3A can comprise an opening 331 at the bottom of thetip. The opening 331 can be utilized for transferring fluids or reagentsin and out of an assay unit 301. In an embodiment, an assay unit 301 asdescribed is or is similar to a pipette tip with the improvement thatthe assay unit 301 comprises a capture surface 311 configured to detectan analyte in a sample.

The tip 301 can be manufactured by an injection-molded process. In anembodiment, the tip 301 is made of a clear polystyrene for use withchemiluminescence assays. As shown in FIG. 3A, an exemplary tip 301comprises a boss (shown as the larger top half of the tip 301), whichcan engage with a housing and can engage, for example, with taperedelements of a fluid transfer device and/or pipetting devices so as toform a pressure-tight seal. Also shown in FIG. 3A, the exemplary tip 301comprises a smaller cylindrical part. In many embodiments, an assaycapture surface is contained within the smaller cylindrical part. Theassay capture surface can be anywhere within the tip 301 or on theoutside of the tip 301. The surface of the tip 301 can be of manygeometries including, but not limited to, tubular, cubic, or pyramidal.In chemiluminescence and fluorescence-based assays, the tip 301 canserve as a convenient means to present the assay product to the assayoptics.

FIG. 3B demonstrates an exemplary sample collection unit 302 comprisinga sample tip 302. The sample tip 302 as shown in FIG. 3B can also beseparate from a sample collection unit 302 and used to transfer samplefrom the sample collection units to other units on a device as describedherein. The sample tip as shown in FIG. 3B comprises a boss 322 asdescribed herein to couple the tip 302 with a housing of a device and afluid transfer device. The sample tip 302 also comprises an opening 332to allow the transfer of fluids or samples in and out of the sample tip.In some embodiments, the sample tip 302 is of the same shape as an assaytip 301. In other embodiments (such as those shown in FIGS. 3A and 3B),the sample tip 302 is a different shape than the assay tip 301.

In an embodiment, one function of a tip is to enable samples and liquidreagents to be brought into contact with the capture surface of theassay unit. The movement can occur by a variety of means including, butnot limited to, capillary action, aspiration, and controlled pumping.The small size of the tips enables rapid control of the requiredtemperature for a chemical reaction. Heat transfer and/or maintenancecan be carried out by simply placing the tip in a temperature controlledblock.

In some embodiments, the tip is able to contain about 1 to 40microliters of fluid. In a further embodiment, the tip is able tocontain about 5 to 25 microliters of fluid. In an embodiment, the tipcontains 20 microliters of fluid. In some instances, a tip can contain 1microliter of fluid or less. In other instances, a tip can contain up to100 microliters.

Where desired, the end of the tip can be blotted onto an absorbentmaterial (for example incorporated into a disposable cartridge) prior tointroduction of the next assay component to avoid contamination with asmall amount of sample and/or reagent. Due to physical forces, anyliquid drawn into a subject tip can be held at any desired location withminimal risk of the liquid draining out, even when held in a verticalorientation.

The assay unit (for example, an assay tip) can be coated with assaycapture reagents prior to use, using similar fluidics as in the assay(for example, controlled capillary or mechanical aspiration).

A capture surface (also referred to herein as a reaction site) can beformed by a binding antibody or other capture reagents bound covalentlyor by adsorption to the assay unit. The surface can then dried andmaintained in dry condition until used in an assay. In an embodiment,there is a reaction site for each analyte to be measured.

In an embodiment, the assay unit can be moved into fluid communicationwith the reagent unit and/or a sample collection unit, such that areagent or sample can interact with a reaction site where bound probescan detect an analyte of interest in the bodily fluid sample. A reactionsite can then provide a signal indicative of the presence orconcentration of the analyte of interest, which can then be detected bya detection device described herein.

In some embodiments, the location and configuration of a reaction siteis an important element in an assay device. Most, if not all, disposableimmunoassay devices have been configured with their capture surface asan integral part of the device.

In one embodiment, a molded plastic assay unit is either commerciallyavailable or can be made by injection molding with precise shapes andsizes. For example, the characteristic dimension can be a diameter of0.05-3 mm or can be a length of 3 to 30 mm. The units can be coated withcapture reagents using method similar to those used to coat microtiterplates but with the advantage that they can be processed in bulk byplacing them in a large vessel, adding coating reagents and processingusing sieves, holders, and the like to recover the pieces and wash themas needed.

The assay unit can offer a rigid support on which a reactant can beimmobilized. The assay unit is also chosen to provide appropriatecharacteristics with respect to interactions with light. For example,the assay unit can be made of a material, such as functionalized glass,Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon, or any one of a widevariety of gels or polymers such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, polypropylene,PMMA, ABS, or combinations thereof. In an embodiment, an assay unitcomprises polystyrene. Other appropriate materials may be used inaccordance with the present invention. A transparent reaction site maybe advantageous. In addition, in the case where there is an opticallytransmissive window permitting light to reach an optical detector, thesurface may be advantageously opaque and/or preferentially lightscattering.

A reactant immobilized at the capture surface can be anything useful fordetecting an analyte of interest in a sample of bodily fluid. Forinstance, such reactants include, without limitation, nucleic acidprobes, antibodies, cell membrane receptors, monoclonal antibodies andantisera reactive with a specific analyte. Various commerciallyavailable reactants such as a host of polyclonal and monoclonalantibodies specifically developed for specific analytes can be used.

One skilled in the art will appreciate that there are many ways ofimmobilizing various reactants onto a support where reaction can takeplace. The immobilization may be covalent or noncovalent, via a linkermoiety, or tethering them to an immobilized moiety. Non-limitingexemplary binding moieties for attaching either nucleic acids orproteinaceous molecules such as antibodies to a solid support includestreptavidin or avidin/biotin linkages, carbamate linkages, esterlinkages, amide, thiolesteri, (N)-functionalized thiourea,functionalized maleimide, amino, disulfide, amide, hydrazone linkages,and among others. In addition, a silyl moiety can be attached to anucleic acid directly to a substrate such as glass using methods knownin the art. Surface immobilization can also be achieved via a Poly-LLysine tether, which provides a charge-charge coupling to the surface.

The assay units can be dried following the last step of incorporating acapture surface. For example, drying can be performed by passiveexposure to a dry atmosphere or via the use of a vacuum manifold and/orapplication of clean dry air through a manifold.

In many embodiments, an assay unit is designed to enable the unit to bemanufactured in a high volume, rapid manufacturing processes. Forexample, tips can be mounted in large-scale arrays for batch coating ofthe capture surface into or onto the tip. In another example, tips canbe placed into a moving belt or rotating table for serial processing. Inyet another example, a large array of tips can be connected to vacuumand/or pressure manifolds for simple processing.

In an embodiment, an assay unit can be operably coupled with a fluidtransfer device. The fluid transfer device can be operated underautomatic control without human interaction. In assay units comprisingtips, the control of the installed height of a disposable liquid tiprelies on the tapered interference attachment of the tip to the liquiddispenser. A fluid transfer device can engage the tip. In someinstances, the immersion length of a tip in liquid to be transferredmust be known to minimize the liquid contact with the outside of the tipwhich may be uncontrolled. In order to couple or adhere a tip to thefluid transfer device a hard stop can be molded at the bottom of thetapered connector which engages the nozzle of the dispenser. An airtight seal can be made by an o-ring that is half way up the taper or inthe flat bottom of the nozzle. By separating the seal function of thetip from the controlled height of the tip both can be separatelyadjusted. The modular device and fluid transfer device can enable manyassays to be performed in parallel.

The reagent units of a device can store reagents that are required toperform a give chemical reaction for detecting a given analyte ofinterest. Liquid reagents can be dispensed into small capsules that canbe manufactured from a variety of materials including, withoutlimitation, plastic such as polystyrene, polyethylene, or polypropylene.In some embodiments, the reagent units are cylindrical cups. Twoexamples of a reagent unit 401, 402 comprising a cup are shown in FIGS.4A and 4B. Where desired, the units 401, 402 fit snugly into cavities ina housing of a device. The units 401, 402 can be sealed on the opensurface to avoid spilling the reagents 411, 412 onboard. In someembodiments, the seal is an aluminized plastic and can be sealed to thecup by thermal bonding. A unit can be of any shape as is necessary tocontain a reagent. For example, a cylindrical shaped reagent unit 401 isshown in FIG. 4A, and the reagent unit contains a liquid reagent 411. Adifferent shaped reagent unit 402 is illustrated in FIG. 4B also containa liquid reagent 412. Both exemplary reagent units 401, 402 compriseoptional slight modifications near the top surface that allow the units401, 402 to fit snugly into a housing of a device as described herein.

In many embodiments of the invention the reagent units are modular. Thereagent unit can be designed to enable the unit to be manufactured in ahigh volume, rapid manufacturing processes. For example, many reagentunits can be filled and sealed in a large-scale process simultaneously.The reagent units can be filled according to the type of assay or assaysto be run by the device. For example, if one user desires differentassays than another user, the reagent units can be manufacturedaccordingly to the preference of each user, without the need tomanufacture an entire device. In another example, reagent units can beplaced into a moving belt or rotating table for serial processing.

In another embodiment, the reagent units are accommodated directly intocavities in the housing of a device. In this embodiment, a seal can bemade onto areas of housing surrounding the units.

Reagents according to the present invention include without limitationwash buffers, enzyme substrates, dilution buffers, conjugates,enzyme-labeled conjugates, DNA amplifiers, sample diluents, washsolutions, sample pre-treatment reagents including additives such asdetergents, polymers, chelating agents, albumin-binding reagents, enzymeinhibitors, enzymes, anticoagulants, red-cell agglutinating agents,antibodies, or other materials necessary to run an assay on a device. Anenzyme-labeled conjugate can be either a polyclonal antibody ormonoclonal antibody labeled with an enzyme that can yield a detectablesignal upon reaction with an appropriate substrate. Non-limitingexamples of such enzymes are alkaline phosphatase and horseradishperoxidase. In some embodiments, the reagents comprise immunoassayreagents. In general, reagents, especially those that are relativelyunstable when mixed with liquid, are confined separately in a definedregion (for example, a reagent unit) within the device.

In some embodiments, a reagent unit contains approximately about 5microliters to about 1 milliliter of liquid. In some embodiments, theunit may contain about 20-200 microliters of liquid. In a furtherembodiment, the reagent unit contains 100 microliters of fluid. In anembodiment, a reagent unit contains about 40 microliters of fluid. Thevolume of liquid in a reagent unit may vary depending on the type ofassay being run or the sample of bodily fluid provided. In anembodiment, the volumes of the reagents do not have to predetermined,but must be more than a known minimum. In some embodiments, the reagentsare initially stored dry and dissolved upon initiation of the assaybeing run on the device.

In an embodiment, the reagent units can be filled using a siphon, afunnel, a pipette, a syringe, a needle, or a combination thereof. Thereagent units may be filled with liquid using a fill channel and avacuum draw channel. The reagent units can be filled individually or aspart of a bulk manufacturing process.

In an embodiment, an individual reagent unit comprises a differentreagent as a means of isolating reagents from each other. The reagentunits may also be used to contain a wash solution or a substrate. Inaddition, the reagent units may be used to contain a luminogenicsubstrate. In another embodiment, a plurality of reagents are containedwithin a reagent unit.

In some instances, the setup of the device enables the capability ofpre-calibration of assay units and the reagent units prior to assemblyof disposables of the subject device.

Systems

In an aspect, a system of the invention comprises a device comprisingassay units and reagent units comprising reagents (both liquid and solidphase reagents). In some embodiments, at least one of the whole device,an assay unit, a reagent unit, or a combination thereof is disposable.In a system of the invention, the detection of an analyte with a deviceis operated by an instrument. In most embodiments, the instrument,device, and method offer an automated detection system. The automateddetection system can be automated based upon a defined protocol or aprotocol provided to the system by a user.

In an aspect, a system for automated detection an analyte in a bodilyfluid sample comprises a device or cartridge, and a detection assemblyor detector for detecting the detectable signal indicative of thepresence or absence of the analyte.

In an embodiment, the user applies a sample (for example, a measured oran unmeasured blood sample) to the device and inserts the device intothe instrument. All subsequent steps are automatic, programmed either bythe instrument (hard wired), the user, a remote user or system, ormodification of the instrument operation according to a identifier (forexample, a bar code or RFID on the device).

Examples of different functions of that can be carried out using asystem of the invention include, but are not limited to, dilution of asample, removal of parts of a sample (for example, red blood cells(RBCs)), reacting a sample in an assay unit, adding liquid reagents tothe sample and assay unit, washing the reagents from the sample andassay unit, and containing liquids during and following use of thedevice. Reagents can be onboard the device in a reagent unit or in areagent unit to assembled onto the device.

An automated system can detect a particular analyte in a biologicalsample (for example, blood) by an enzyme-linked immunosorbent assay(ELISA). The system is amenable to multiplexing and is particularlysuited for detecting an analyte of interest present in a small volume ofa whole blood sample (for example, 20 microliters or less). The systemcan also detect analytes in different dilutions of a single sample,allowing different sensitivities to be tested on the same device, whendesired. All reagents, supplies, and wastes can be contained on thedevice of the system.

In use, a sample from a subject is applied to the assembled device andthe device is inserted into an instrument. In an embodiment, aninstrument can begin processing the sample by some combination ofremoval of red cells (blood sample), dilution of the sample, andmovement the sample to the assay unit. In an embodiment with multiplexedassays, a plurality of assay units are used and a portion of the sampleis moved to individual assay units in sequence or in parallel. Assayscan then be performed by a controlled sequence of incubations andapplications of reagents to the capture surfaces.

An exemplary fluid transfer device is comprised of any componentrequired to perform and/or read the assay. Example of componentsinclude, but are not limited to, pumps to aspirate and eject accuratelyknown fluid volumes from wells or units of the device, at least onetranslational stage for improving the precision and accuracy of themovement within the system, a detector to detect an analyte in an assayunit, and temperature regulation means to provide a regulatedtemperature environment for incubation of assays. In an embodiment ofthe invention, the instrument controls the temperature of the device. Ina further embodiment, the temperature is in the range of about 30-40degrees Celsius. In some embodiments, the temperature control by thesystem can comprise active cooling. In some instances, the range oftemperature is about 0-100 degrees Celsius. For example, for nucleicacid assays, temperatures up to 100 degrees Celsius can be achieved. Inan embodiment, the temperature range is about 15-50 degrees Celsius. Atemperature control unit of the system can comprise a thermoelectricdevice, such as a Peltier device.

Cartridges, devices, and systems as described herein can offer manyfeatures that are not available in existing POC systems or integratedanalysis systems. For example, many POC cartridges rely on a closedfluidic system or loop to handle small volumes of liquid in an efficientmanner. The cartridges and fluidic devices described herein can haveopen fluid movement between units of the cartridge. For example, areagent can be stored in a unit, a sample in a sample collection unit, adiluent in a diluent unit, and the capture surface can be in an assayunit, wherein in one state of cartridge, none of the units are in fluidcommunication with any of the other units. Using a fluid transfer deviceor system as described herein, the units do not have to be in fluidcommunication with each other in a state. The units can be movablerelative to each other in order to bring some units into fluidcommunication. For example, a fluid transfer device can comprise a headthat engages an assay unit and moves the assay unit into fluidiccommunication with a reagent unit.

The devices and systems herein can provide an effective means for highthroughput and real-time detection of analytes present in a bodily fluidfrom a subject. The detection methods may be used in a wide variety ofcircumstances including identification and quantification of analytesthat are associated with specific biological processes, physiologicalconditions, disorders or stages of disorders. As such, the systems havea broad spectrum of utility in, for example, drug screening, diseasediagnosis, phylogenetic classification, parental and forensicidentification, disease onset and recurrence, individual response totreatment versus population bases, and monitoring of therapy. Thesubject devices and systems are also particularly useful for advancingpreclinical and clinical stage of development of therapeutics, improvingpatient compliance, monitoring ADRs associated with a prescribed drug,developing individualized medicine, outsourcing blood testing from thecentral laboratory to the home or on a prescription basis, andmonitoring therapeutic agents following regulatory approval. The devicesand systems can provide a flexible system for personalized medicine.Using the same system, a device can be changed or interchanged alongwith a protocol or instructions to a programmable processor of thesystems to perform a wide variety of assays as described. The systemsand devices herein offer many features of a laboratory setting in adesk-top or smaller size automated instrument.

In some embodiments a patient may be provided with a plurality ofdevices to be used for detecting a variety of analytes. A subject may,for example, use different fluidic devices on different days of theweek. In some embodiments the software on the external deviceassociating the identifier with a protocol may include a process tocompare the current day with the day the fluidic device is to be usedbased on a clinical trial for example. In another embodiment, thepatient is provided different reagent units and assay units that can befit into a housing of a device interchangeably. In yet anotherembodiment, as described the patient does not need a new device for eachday of testing, but rather, the system can be programmed or reprogrammedby downloading new instructions from, e.g. an external device such as aserver. If for example, the two days of the week are not identical, theexternal device can wirelessly send notification to the subject usingany of the methods described herein or known in the art to notify themof the proper device and/or proper instructions for the system. Thisexample is only illustrative and can easily be extended to, for example,notifying a subject that a fluidic device is not being used at thecorrect time of day.

For example, a cartridge as illustrated in FIG. 1 can comprise a varietyof assay units and reagent units. The assay units can comprise a capturesurface according to an analyte to be detected. The assay units can thenbe assembled with the rest of the device in a just-in-time fashion. Inmany prior art POC devices, the capture surface is integral to thedevice and if the capture surface is incorrect or not properly formed,the whole device is bad. Using a device as described herein, the capturesurface and/or assay unit can be individually quality controlled andcustomized independently of the reagent units and the housing of thedevice.

Reagent units can be filled with a variety of reagents in a similarjust-in-time fashion. This provides flexibility of the device beingcustomizable. In addition, the reagent units can be filled withdifferent volumes of reagents without affecting the stability of adevice or the chemical reactions to be run within the device. Coupledwith a system as described with a fluid transfer device, the devices andunits described herein offer flexibility in the methods and protocols ofthe assays to be run. For example, a batch of similar devices containingthe same reagents can be given to a patient pool for a clinical trial.Half way through the clinical trial, a user identifies that the assaycould be optimized by changing the dilution of the sample and the amountof reagent provided to the assay unit. As provided herein, the assay canbe changed or optimized by only changing the instructions to aprogrammable processor of the fluid transfer device. For example, thebatch of cartridges in the patient pool had excess diluent loaded on thecartridge. The new protocol demands four times as much diluent as theprevious protocol. Due to the methods and systems provided herein, theprotocol can be changed at a central server and sent to all the systemsfor executing the methods with the devices without having to provide newdevices to the patient pool. In other words, a POC device and system asdescribed herein can offer much of the flexibility of a standardlaboratory practice where excess reagents and often excess sample areoften available.

In some instances, wherein the units of the cartridge are separate,devices and systems provide flexibility in construction of the systemsdescribed herein. For example, a cartridge can be configured to run 8assays using an array of assay units and an array of reagent units. Dueto the features of the cartridge as described herein, the same housing,or a housing of the same design can be used to manufacture a cartridgewith up to 8 different assays than the previous cartridge. Thisflexibility is difficult to achieve in many current POC device designsbecause of the closed systems and fluid channels, and therefore thedevices may not be modular or as easy to assemble as described.

Currently, a need exists for the detecting more than one analyte wherethe analytes are present in widely varying concentration range, forexample, one analyte is in the pg/ml concentration range and another isin the ug/ml concentration range. The system as described herein has theability to simultaneously assay analytes that are present in the samesample in a wide concentration range. Another advantage for being ableto detect concentrations of different analytes present in a wideconcentration range is the ability to relate the ratios of theconcentration of these analytes to safety and efficacy of multiple drugsadministered to a patient. For example, unexpected drug-druginteractions can be a common cause of adverse drug reactions. Areal-time, concurrent measurement technique for measuring differentanalytes would help avoid the potentially disastrous consequence ofadverse drug-drug interactions.

Being able to monitor the rate of change of an analyte concentrationand/or or concentration of PD or PK markers over a period of time in asingle subject, or performing trend analysis on the concentration, ormarkers of PD, or PK, whether they are concentrations of drugs or theirmetabolites, can help prevent potentially dangerous situations. Forexample, if glucose were the analyte of interest, the concentration ofglucose in a sample at a given time as well as the rate of change of theglucose concentration over a given period of time could be highly usefulin predicting and avoiding, for example, hypoglycemic events. Such trendanalysis has widespread beneficial implications in drug dosing regimen.When multiple drugs and their metabolites are concerned, the ability tospot a trend and take proactive measures is often desirable.

Accordingly, the data generated with the use of the subject fluidicdevices and systems can be utilized for performing a trend analysis onthe concentration of an analyte in a subject.

Often, 8 assays on the same cartridge may require different dilutions orpre-treatments. The range of dilution can be substantial between assays.Many current POC devices offer a limited range of dilution and thereforea limited number of assays that can be potentially carried out on thePOC device. However, a system and/or cartridge as described herein canoffer a large range of dilutions due to the ability of to seriallydilute a sample. Therefore, a large number of potential assays can beperformed on a single cartridge or a plurality of cartridges withoutmodifying the detector or reading instrument for the assays.

In an example, a system as provided herein is configured to run multiple(e.g., five or more) different target analyte detection assays. In orderto bring the expected analyte concentration within the range ofdetection of an immunoassay as described herein and commonly used in thePOC field, a sample must be diluted e.g., 3:1, 8:1, 10:1, 100:1, and2200:1, to run each of the five assays. Because the fluid transferdevice is able to hold and move fluid within the device, serialdilutions can be performed with a system as described herein to achievethese five different dilutions and detect all five different targetanalytes. As described above, the protocol for performing the assays isalso capable of being adjusted without modifying the device or thesystem.

In a laboratory setting with traditional pipetting, typically largervolumes of sample are used than in a POC setting. For example, alaboratory may analyze a blood sample withdrawn from the arm of apatient in a volume in the milliliter range. In a POC setting, manydevices and users demand that the process is fast, easy and/or minimallyinvasive, therefore, small samples (on the order of a volume in themicroliter range) such as one obtained by a fingerstick) are typicallyanalyzed by a POC device. Because of the difference in sample, currentPOC devices can lose flexibility in running an assay that is afforded ina laboratory setting. For example, to run multiple assays from a sample,a certain minimum volume can be required for each assay to allow foraccurate detection of an analyte, therefore putting some limits on adevice in a POC setting.

In another example, a system and/or fluid transfer device as describedherein provides a great deal of flexibility. For example, the fluidtransfer device can be automated to move an assay unit, an assay tip, oran empty pipette from one unit of the device to a separate unit of thedevice, not in fluid communication with each other. In some instances,this can avoid cross-contamination of the units of a device asdescribed. In other instances, it allows for the flexibility of movingseveral fluids within a device as described into contact with each otheraccording to a protocol or instructions. For example, a cartridgecomprising 8 different reagents in 8 different reagent units can beaddressed and engaged by a fluid transfer device in any order orcombination as is instructed by a protocol. Therefore, many differentsequences can be run for any chemical reaction to run on the device.Without changing the volume of the reagents in the cartridge or the typeof reagents in the cartridge, the assay protocol can be different ormodified without the need for a second cartridge or a second system.

For example, a user orders a cartridge with a specific type of capturesurface and specific reagents to run an assay to detect an analyte (forexample, C-reactive protein (CRP)) in a sample. The protocol the useroriginally planned for may require 2 washing steps and 3 dilution steps.After the user has received the device and system, the user has decidedthat the protocol should actually have 5 washing steps and only 1dilution step. The devices and systems herein can allow the flexibilityfor this change in protocol without having to reconfigure the device orthe system. In this example, only a new protocol or set of instructionsare needed to be sent to the programmable processor of the system or thefluid transfer device.

In another example, a system as provided herein is configured to runfive different target analyte detection assays, wherein each assay needsto be incubated at a different temperature. In many prior art POCdevices, incubation of multiple assays at different temperatures is adifficult task because the multiple assays are not modular and thecapture surfaces cannot be moved relative to the heating device. In asystem as described herein, wherein an individual assay unit isconfigured to run a chemical reaction, an individual assay unit can beplace in an individual heating unit. In some embodiments, a systemcomprises a plurality of heating units. In some instances, a systemcomprises at least as many heating units as assay units. Therefore, aplurality of assays can be run as a plurality of temperatures.

Systems and devices as described herein can also provide a variety ofquality control measures not previously available with many prior artPOC devices. For example, because of the modularity of a device, theassay units and reagents units can be quality controlled separately fromeach other and/or separately from the housing and/or separately from asystem or fluid transfer device. Exemplary methods and systems ofquality control offered by the systems and devices herein are described.

A system as described can run a variety of assays, regardless of theanalyte being detected from a bodily fluid sample. A protocol dependenton the identity of the device may be transferred from an external devicewhere it can be stored to a reader assembly to enable the readerassembly to carry out the specific protocol on the device. In someembodiments, the device has an identifier (ID) that is detected or readby an identifier detector described herein. The identifier detector cancommunicate with a communication assembly via a controller whichtransmits the identifier to an external device. Where desired, theexternal device sends a protocol stored on the external device to thecommunication assembly based on the identifier. The protocol to be runon the system may comprise instructions to the controller of the systemto perform the protocol, including but not limited to a particular assayto be run and a detection method to be performed. Once the assay isperformed by the system, a signal indicative of an analyte in the bodilyfluid sample is generated and detected by a detection assembly of thesystem. The detected signal may then be communicated to thecommunications assembly, where it can be transmitted to the externaldevice for processing, including without limitation, calculation of theanalyte concentration in the sample.

In some embodiments, the identifier may be a bar code identifier with aseries of black and white lines, which can be read by an identifierdetector such as a bar code reader, which are well known. Otheridentifiers could be a series of alphanumerical values, colors, raisedbumps, or any other identifier which can be located on a device and bedetected or read by an identifier detector. The identifier detector mayalso be an LED that emits light which can interact with an identifierwhich reflects light and is measured by the identifier detector todetermine the identity of a device. In some embodiments the identifiermay comprise a storage or memory device and can transmit information toan identification detector. In some embodiments a combination oftechniques may be used. In some embodiments, the detector is calibratedby used of an optical source, such as an LED.

In an example, a bodily fluid sample can be provided to a device, andthe device can be inserted into a system. In some embodiments the deviceis partially inserted manually, and then a mechanical switch in thereader assembly automatically properly positions the device inside thesystem. Any other mechanism known in the art for inserting a disk orcartridge into a system may be used. In some embodiments, manualinsertion may be required.

In some embodiments a method of automatically selecting a protocol to berun on a system comprises providing a device comprising an identifierdetector and an identifier; detecting the identifier; transferring saididentifier to an external device; and selecting a protocol to be run onthe system from a plurality of protocols on said external deviceassociated with said identifier.

In an aspect, a system for automated detection of a plurality ofanalytes in a bodily fluid sample is disclosed that comprises: a fluidicdevice (such as those described herein) comprising: a sample collectionunit configured to contain the bodily fluid sample; an array of assayunits, wherein an individual assay unit of said array of assay units isconfigured to run a chemical reaction that yields a signal indicative ofan individual analyte of said plurality of analytes being detected; andan array of reagent units, wherein an individual reagent unit of saidarray of reagent units contains a reagent. The system further comprisesa fluid transfer device comprising a plurality of heads, wherein anindividual head of the plurality of heads is configured to engage theindividual assay unit, and wherein said fluid transfer device comprisesa programmable processor configured to direct fluid transfer of thebodily fluid sample from the sample collection unit and the reagent fromthe individual reagent unit into the individual assay unit. For example,an individual assay unit comprises a reagent and is configured is to runa chemical reaction with that reagent.

In some instances, the configuration of the processor to direct fluidtransfer effects a degree of dilution of the bodily fluid sample in thearray of assay units to bring signals indicative of the plurality ofanalytes being detected within a detectable range, such that saidplurality of analytes are detectable with said system. In an example,the bodily fluid sample comprises at least two analytes that are presentat concentrations that differ by at least 2, 5, 10, 15, 50, or 100orders of magnitude. In an example the bodily fluid sample is a singledrop of blood. In an embodiment, the concentrations of at least twoanalytes present in a sample differs by up to 10 orders of magnitude(for example, a first analyte is present at 0.1 pg/mL and a secondanalyte is present at 500 ug/mL. In another example, some proteinanalytes are found at concentrations of greater than 100 mg/mL, whichcan extend the range of interest to about twelve orders of magnitude.

A degree of dilution of the bodily fluid sample can bring the signalsindicative of the at least two analytes within the detectable range. Inmany instances, a system further comprises a detector, such as aphotomultiplier (PMT). With a photomultiplier, for example, a detectablerange of the detector can be about 10 to about 10 million counts persecond. Each count corresponds to a single photon. In some instances,PMTs are not 100% efficient and the observed count rate may be slightlylower than, but still close to, the actual number of photons reachingthe detector per unit time. In some instances, counts are measured inabout ten intervals of about one second and the results are averaged. Insome embodiments, ranges for assays are 1000-1,000,000 counts per secondwhen using a PMT as a detector. In some instances, count rates as low as100 per second and count rates as high as 10,000,000 are measurable. Thelinear response range of PMTs (for example, the range where count rateis directly proportional to number of photons per unit time) can beabout 1000-3,000,000 counts per second. In an example, an assay has adetectable signal on the low end of about 200-1000 counts per second andon the high end of about 10,000-2,000,000 counts per second. In someinstances for protein biomarkers, the count rate is directlyproportional to alkaline phosphatase bound to the capture surface andalso directly proportional to the analyte concentration. Other exemplarydetectors include avalanche photodiodes, avalanche photodiode arrays,CCD arrays, super-cooled CCD arrays. Many other detectors have an outputthat is digital and generally proportional to photons reaching thedetector. The detectable range for exemplary detectors can be suitableto the detector being used.

An individual head of a fluid transfer device can be configured toadhere to the individual assay unit. The fluid transfer device can be apipette, such as an air-displacement pipette. The fluid transfer devicecan be automated. For example, a fluid transfer device can furthercomprise a motor in communication with a programmable processor and themotor can move the plurality of heads based on a protocol from theprogrammable processor. As described, an individual assay unit can be apipette tip, for example, a pipette tip with a capture surface orreaction site.

Often times, in a POC device, such as the systems and devices describedherein, the dilution factor must be estimated and reasonably precise.For example, in environments where non-expert users operate the systemthere needs to be ways of ensuring a dilution of a sample.

As described herein, a fluid transfer device can affect a degree ofdilution of a sample to provide accurate assay results. For example, aprogrammable fluid transfer device can be multi-headed) to dilute orserially dilute samples as well as provide mixing of a sample anddiluent. A fluid transfer device can also provide fluid movement in POCdevices.

As described, the systems and devices herein can enable many features ofthe flexibility of laboratory setting in a POC environment. For example,samples can be collected and manipulated automatically in a table topsize or smaller device or system. A common issue in POC devices isachieving different dilution ranges when conducting a plurality ofassays, wherein the assays may have significantly different sensitivityor specificity. For example, there may be two analytes in a sample, butone analyte has a high concentration in the sample and the other analytehas a very low concentration. As provided, the systems and devicesherein can dilute the sample to significantly different levels in orderto detect both analytes. For example, if the analyte is in a highconcentration, a sample can be serially diluted to the appropriatedetection range and provided to a capture surface for detection. In thesame system or device, a sample with an analyte in a low concentrationmay not need to be diluted. In this manner, the assay range of the POCdevices and systems provided herein can be expanded from many of thecurrent POC devices.

A fluid transfer device can be part of a system that is a bench-topinstrument. The fluid transfer device can comprise a plurality of heads.Any number of heads as is necessary to detect a plurality of analytes ina sample is envisioned for a fluid transfer device of the invention. Inan example, a fluid transfer device has about eight heads mounted in aline and separated by a distance. In an embodiment, the heads have atapered nozzle that engages by press fitting with a variety of tips,such as assay unit or sample collection units as described herein. Thetips can have a feature that enables them to be removed automatically bythe instrument and disposed into in a housing of a device as describedafter use. In an embodiment, the assay tips are clear and transparentand can be similar to a cuvette within which an assay is run that can bedetected by an optical detector such as a photomultiplier tube.

In an example, the programmable processor of a system can compriseinstructions or commands and can operate a fluid transfer deviceaccording to the instructions to transfer liquid samples by eitherwithdrawing (for drawing liquid in) or extending (for expelling liquid)a piston into a closed air space. Both the volume of air moved and thespeed of movement can be precisely controlled, for example, by theprogrammable processor.

Mixing of samples (or reagents) with diluents (or other reagents) can beachieved by aspirating components to be mixed into a common tube andthen repeatedly aspirating a significant fraction of the combined liquidvolume up and down into a tip. Dissolution of reagents dried into a tubecan be done is similar fashion. Incubation of liquid samples andreagents with a capture surface on which is bound a capture reagent (forexample an antibody) can be achieved by drawing the appropriate liquidinto the tip and holding it there for a predetermined time. Removal ofsamples and reagents can be achieved by expelling the liquid into areservoir or an absorbent pad in a device as described. Another reagentcan then be drawn into the tip according to instructions or protocolfrom the programmable processor.

In an example as illustrated in FIG. 11, a liquid 1111 previously in atip 1101 can leave a thin film 1113 within the tip 1101 when expelled.Therefore, a system can use the action of the leading (for exampleuppermost) portion of the next liquid 1112 to scour the previouslypresent liquid 1111 from the tip 1101. The portion of the subsequentliquid contaminated with the liquid previously present 1113 can be heldwithin the top of the tip 1101 where it does not continue to interactwith the capture surface 1102. The capture surface 1102 can be in adefined area of the tip 1101 such that the previous liquid 1111 does notreact with the capture surface 1102, for example as shown in FIG. 11,the capture surface 1102 occupies a defined portion of the cylindricalpart of the tip 1101 not extending all the way up to the boss of thetip. In many instances, incubation time is short (for example 10minutes) and separation of the contaminated zone of liquid is relativelylarge (>1 mm) so diffusion or the active components of the contaminatedportion of liquid 1113 does not occur rapidly enough react with thecapture surface 1102 during the incubation. For many high sensitivityassays, there is a requirement to remove one reagent or wash the capturesurface (for example, a detector antibody which is labeled with theassay signal generator). In an example, a fluid transfer device of asystem described herein can provide washing by adding further removaland aspiration cycles of fluid transfer, for example, using a washreagent. In an example, four wash steps demonstrated that the unbounddetector antibody in contact with the capture surface is reduced by afactor of better than 10⁶-fold. Any detector antibody non-specificallybound to the capture surface (highly undesirable) can also be removedduring this wash process.

Extension of the range of an assay can be accomplished by dilution ofthe sample. In POC assay systems using disposable cartridges containingthe diluent there is often a practical limit to the extent of dilution.For example, if a small blood sample is obtained by fingerstick (forexample, about 20 microliters) is to be diluted and the maximum volumeof diluent that can be placed in a tube is 250 microliters, thepractical limit of dilution of the whole sample is about 10-fold. In anexample herein, a system can aspirate a smaller volume of the sample(for example about 2 microliters) making the maximum dilution factorabout 100-fold. For many assays, such dilution factors are acceptablebut for an assay like that of CRP (as described in the examples herein)there is a need to dilute the sample much more. Separation-based ELISAassays can have an intrinsic limitation in thee capacity of the capturesurface to bind the analyte (for example about a few hundred ng/ml for atypical protein analyte). Some analytes are present in blood at hundredsof micrograms/ml. Even when diluted by 100-fold, the analyteconcentration may be outside the range of calibration. In an exemplaryembodiment of a system, device, and fluid transfer device herein,multiple dilutions can be achieved by performing multiple fluidtransfers of the diluent into an individual assay unit or samplecollection unit. For example, if the concentration of an analyte is veryhigh in a sample as described above, the sample can be diluted multipletimes until the concentration of the analyte is within an acceptabledetection range. The systems and methods herein can provide accuratemeasurements or estimations of the dilutions in order to calculate theoriginal concentration of the analyte.

In an embodiment, a system herein can move a liquid sample and move anassay unit. A system can comprise a heating block and a detector. Inorder to move a liquid sample, a system may provide aspiration-,syringe-, or pipette-type action. In an exemplary embodiment, a fluidtransfer device for moving a liquid sample is a pipette and pipette headsystem. The number of pipette devices required by the system can beadjusted according to the type of analyte to be detected and the numberof assays being run. The actions performed by the pipette system can beautomated or operated manually by a user.

FIG. 5 demonstrates an example of a fluid transfer device 520 and system500 as described herein. The fluid transfer device system can move eightdifferent or identical volumes of liquid simultaneously using the eightdifferent heads 522. For example, the cartridge (or device as describedherein) 510 comprises eight assay units 501. Individual assay units 501are configured according to the type of assay to be run within the unit501. Individual assay units 501 may require a certain volume of sample.An individual head 522 can be used to distribute a proper amount ofsample to an individual assay unit 501. In this example, each head 522corresponds to an addressed individual assay unit 501.

The fluid transfer device mechanism 520 can also be used to distributereagents from the reagent units. Different types of reagents include aconjugate solution, a wash solution, and a substrate solution. In anautomated system, the stage 530 on which the device 510 sits can bemoved to move the device 510 relative to the positioning of the assayunits 501 and heads 522 and according to the steps necessary to completean assay as demonstrated in FIG. 5. Alternatively, the heads 522 andtips 501 or the fluid transfer device 520 can be moved relative to theposition of the device 510.

In some embodiments, a reagent is provided in dry form and rehydratedand/or dissolved during the assy. Dry forms include lyophilizedmaterials and films coated on surfaces.

A system can comprise a holder or engager for moving the assay units ortips. An engager may comprise a vacuum assembly or an assembly designedto fit snugly into a boss of an assay unit tip. For example, a means formoving the tips can be moved in a manner similar to the fluid transferdevice heads. The device can also be moved on a stage according to theposition of an engager or holder.

In an embodiment, an instrument for moving the tips is the same as aninstrument for moving a volume of sample, such as a fluid transferdevice as described herein. For example, a sample collection tip can befit onto a pipette head according to the boss on the collection tip. Thecollection tip can then be used to distribute the liquid throughout thedevice and system. After the liquid has been distributed, the collectiondip can be disposed, and the pipette head can be fit onto an assay unitaccording to the boss on the assay unit. The assay unit tip can then bemoved from reagent unit to reagent unit, and reagents can be distributedto the assay unit according to the aspiration- or pipette-type actionprovided by the pipette head. The pipette head can also perform mixingwithin a collection tip, assay unit, or reagent unit by aspiration- orsyringe-type action.

A system can comprise a heating block for heating the assay or assayunit and/or for control of the assay temperature. Heat can be used inthe incubation step of a assay reaction to promote the reaction andshorten the duration necessary for the incubation step. A system cancomprise a heating block configured to receive an assay unit of theinvention. The heating block can be configured to receive a plurality ofassay units from a device of the invention. For example, if 8 assays aredesired to be run on a device, the heating block can be configured toreceive 8 assay units. In some embodiments, assay units can be movedinto thermal contact with a heating block using the means for moving theassay units. The heating can be performed by a heating means known inthe art.

An exemplary system 600 as described herein is demonstrated in FIG. 6.The system 600 comprises a translational stage 630 onto which a device610 (or cartridge in this example) is placed either manually orautomatically or a combination of both. The system 600 also comprises aheating block 640 that can be aligned with the assay units 611 of thedevice 610. As shown in FIG. 6, the device 610 comprises a series of 8assay units 611 and multiple corresponding reagent units 612, and theheating block 640 also comprises an area 641 for at least 8 units to beheated simultaneously. Each of the heating areas 641 can provide thesame or different temperatures to each individual assay unit 611according to the type of assay being run or the type of analyte beingdetected. The system 600 also comprises a detector (such as aphotomultiplier tube) 650 for detection of a signal from an assay unit611 representative of the detection of an analyte in a sample.

In an embodiment, a sensor is provided to locate an assay unit relativeto a detector when an assay is detected.

In an embodiment, the detector is a reader assembly housing a detectionassembly for detecting a signal produced by at least one assay on thedevice. The detection assembly may be above the device or at a differentorientation in relation to the device based on, for example, the type ofassay being performed and the detection mechanism being employed. Thedetection assembly can be moved into communication with the assay unitor the assay unit can be moved into communication with the detectionassembly.

In many instances, an optical detector is provided and used as thedetection device. Non-limiting examples include a photodiode,photomultiplier tube (PMT), photon counting detector, avalanche photodiode, or charge-coupled device (CCD). In some embodiments a pin diodemay be used. In some embodiments a pin diode can be coupled to anamplifier to create a detection device with a sensitivity comparable toa PMT. Some assays may generate luminescence as described herein. Insome embodiments chemiluminescence is detected. In some embodiments adetection assembly could include a plurality of fiber optic cablesconnected as a bundle to a CCD detector or to a PMT array. The fiberoptic bundle could be constructed of discrete fibers or of many smallfibers fused together to form a solid bundle. Such solid bundles arecommercially available and easily interfaced to CCD detectors.

A detector can also comprise a light source, such as a bulb or lightemitting diode (LED). The light source can illuminate an assay in orderto detect the results. For example, the assay can be a fluorescenceassay or an absorbance assay, as are commonly used with nucleic acidassays. The detector can also comprise optics to deliver the lightsource to the assay, such as a lens or fiber optics.

In some embodiments, the detection system may comprise non-opticaldetectors or sensors for detecting a particular parameter of a subject.Such sensors may include temperature, conductivity, potentiometricsignals, and amperometric signals, for compounds that are oxidized orreduced, for example, O₂, H₂O₂, and I₂, or oxidizable/reducible organiccompounds.

A device and system may, after manufacturing, be shipped to the enduser, together or individually. The device or system of the inventioncan be packaged with a user manual or instructions for use. In anembodiment, the system of the invention is generic to the type of assaysrun on different devices. Because components of the device can bemodular, a user may only need one system and a variety of devices orassay units or reagent units to run a multitude of assays in apoint-of-care environment. In this context, a system can be repeatedlyused with multiple devices, and it may be necessary to have sensors onboth the device and the system to detect such changes during shipping,for example. During shipping, pressure or temperature changes can impactthe performance of a number of components of the present system, and assuch a sensor located on either the device or system can relay thesechanges to, for example, the external device so that adjustments can bemade during calibration or during data processing on the externaldevice. For example, if the temperature of a fluidic device is changedto a certain level during shipping, a sensor located on the device coulddetect this change and convey this information to the system when thedevice is inserted into the system by the user. There may be anadditional detection device in the system to perform these tasks, orsuch a device may be incorporated into another system component. In someembodiments information may be wirelessly transmitted to either thesystem or the external device, such as a personal computer or atelevision. Likewise, a sensor in the system can detect similar changes.In some embodiments, it may be desirable to have a sensor in theshipping packaging as well, either instead of in the system componentsor in addition thereto. For example, adverse conditions that wouldrender an assay cartridge or system invalid that can be sensed caninclude exposure to a temperature higher than the maximum tolerable orbreach of the cartridge integrity such that moisture penetration.

In an embodiment, the system comprises a communication assembly capableof transmitting and receiving information wirelessly from an externaldevice. Such wireless communication may be Bluetooth or RTM technology.Various communication methods can be utilized, such as a dial-up wiredconnection with a modem, a direct link such as a T1, ISDN, or cableline. In some embodiments, a wireless connection is established usingexemplary wireless networks such as cellular, satellite, or pagernetworks, GPRS, or a local data transport system such as Ethernet ortoken ring over a local area network. In some embodiments theinformation is encrypted before it is transmitted over a wirelessnetwork. In some embodiments the communication assembly may contain awireless infrared communication component for sending and receivinginformation. The system may include integrated graphic cards tofacilitate display of information.

In some embodiments the communication assembly can have a memory orstorage device, for example localized RAM, in which the informationcollected can be stored. A storage device may be required if informationcan not be transmitted at a given time due to, for example, a temporaryinability to wirelessly connect to a network. The information can beassociated with the device identifier in the storage device. In someembodiments the communication assembly can retry sending the storedinformation after a certain amount of time.

In some embodiments an external device communicates with thecommunication assembly within the reader assembly. An external devicecan wirelessly or physically communicate with a system, but can alsocommunicate with a third party, including without limitation a patient,medical personnel, clinicians, laboratory personnel, or others in thehealth care industry.

An exemplary method and system is demonstrated in FIG. 7. In the exampleof FIG. 7, a patient delivers a blood sample to a device as describedherein and then the device is inserted into a reader, wherein the readercan be desktop system capable of reading an analyte in the blood sample.The reader can be a system as described herein. The reader can be abench-top or desk-top system and can be capable of reading a pluralityof different devices as described herein. The reader or system iscapable of carrying out a chemical reaction and detecting or reading theresults of the chemical reaction. In the example in FIG. 7, a reader isautomated according to a protocol sent from an external device (forexample, a server comprising a user interface). A reader can also sendthe results of the detection of the chemical reaction to the server anduser interface. In an exemplary system, the user (for example, medicalpersonnel such as a physician or researcher) can view and analyze theresults as well as decide or develop the protocol used to automate thesystem. Results can also be stored locally (on the reader) or on theserver system. The server can also host patient records, a patientdiary, and patient population databases.

FIG. 8 illustrates the process flow of building a system for assessingthe medical condition of a subject. The patient inputs personal data andor measurements from a device, reader, and/or system as described hereininto a database as may be present on a server as described. The systemcan configured to display the personal data on a patient stationdisplay. In some embodiments, the patient station display is interactiveand the patient can modify inputted data. The same or a differentdatabase contains data from other subjects with a similar medicalcondition. Data from the other subjects can be historical data frompublic or private institutions. Data from other subjects may also beinternal data from a clinical study.

FIG. 8 also illustrates the flow of data from reader collection datathat includes the data from the subject to a server that is connectedover a public network. The server can manipulate the data or can justprovide the data to a user station. The patient data may also be inputto the server separately from the data pertaining to a medical conditionthat is stored in a database. FIG. 8 also demonstrates a user stationdisplay and the flow of information to medical personnel or a user. Forexample, using the exemplary process flow of FIG. 8, a patient at homecan input a bodily fluid sample into a cartridge of the invention asdescribed herein and place it in a system or reader as described herein.The patient can view the data from the system at a patient stationdisplay and/or modify or input new data into the process flow. The datafrom the patient can then travel over a public network, such as theinternet, for example, in an encrypted format, to a server comprising anetwork interface and a processor, wherein the server is located at acentral computing hub or in a clinical trial center. The server can usemedical condition data to manipulate and understand the data from theuser and then send the results over a public network as described to auser station. The user station can be in a medical office or laboratoryand have a user station display to display the results of the assay andmanipulation of the patient data to the medical personnel. In thisexample, the medical personnel can receive results and analysis of asample from a patient from a test that the patient administered in analternate location such as the patient's home. Other embodiments andexample of systems and components of systems are described herein.

In some embodiments the external device can be a computer system,server, or other electronic device capable of storing information orprocessing information. In some embodiments the external device includesone or more computer systems, servers, or other electronic devicescapable of storing information or processing information. In someembodiments an external device may include a database of patientinformation, for example but not limited to, medical records or patienthistory, clinical trial records, or preclinical trial records. Anexternal device can store protocols to be run on a system which can betransmitted to the communication assembly of a system when it hasreceived an identifier indicating which device has been inserted in thesystem. In some embodiments a protocol can be dependent on a deviceidentifier. In some embodiments the external device stores more than oneprotocol for each device. In other embodiments patient information onthe external device includes more than one protocol. In some instances,the external server stores mathematical algorithms to process a photoncount sent from a communication assembly and in some embodiments tocalculate the analyte concentration in a bodily fluid sample.

In some embodiments, the external device can include one or more serversas are known in the art and commercially available. Such servers canprovide load balancing, task management, and backup capacity in theevent of failure of one or more of the servers or other components ofthe external device, to improve the availability of the server. A servercan also be implemented on a distributed network of storage andprocessor units, as known in the art, wherein the data processingaccording to the present invention reside on workstations such ascomputers, thereby eliminating the need for a server.

A server can includes a database and system processes. A database canreside within the server, or it can reside on another server system thatis accessible to the server. As the information in a database maycontains sensitive information, a security system can be implementedthat prevents unauthorized users from gaining access to the database.

One advantage of some of the features described herein is thatinformation can be transmitted from the external device back to not onlythe reader assembly, but to other parties or other external devices, forexample without limitation, a PDA or cell phone. Such communication canbe accomplished via a wireless network as disclosed herein. In someembodiments a calculated analyte concentration or other patientinformation can be sent to, for example but not limited to, medicalpersonnel or the patient.

Accordingly, the data generated with the use of the subject devices andsystems can be utilized for performing a trend analysis on theconcentration of an analyte in a subject.

Another advantage as described herein is that assay results can besubstantially immediately communicated to any third party that maybenefit from obtaining the results. For example, once the analyteconcentration is determined at the external device, it can betransmitted to a patient or medical personnel who may need to takefurther action. The communication step to a third party can be performedwirelessly as described herein, and by transmitting the data to a thirdparty's hand held device, the third party can be notified of the assayresults virtually anytime and anywhere. Thus, in a time-sensitivescenario, a patient may be contacted immediately anywhere if urgentmedical action may be required.

By detecting a device based on an identifier associated with a fluidicdevice after it is inserted in the system, the system allows for fluidicdevice-specific protocols to be downloaded from an external device andrun. In some embodiments an external device can store a plurality ofprotocols associated with the system or associated with a particularpatient or group of patients. For example, when the identifier istransmitted to the external device, software on the external device canobtain the identifier. Once obtained, software on the external device,such as a database, can use the identifier to identify protocols storedin the database associated with the identifier. If only one protocol isassociated with the identifier, for example, the database can select theprotocol and software on the external device can then transmit theprotocol to the communication assembly of the system. The ability to useprotocols specifically associated with a device allows for any componentof a device of the invention to be used with a single system, and thusvirtually any analyte of interest can be detected with a single system.

In some embodiments multiple protocols may be associated with a singleidentifier. For example, if it is beneficial to detect from the samepatient an analyte once a week, and another analyte twice a week,protocols on the external device associated with the identifier can alsoeach be associated with a different day of the week, so that when theidentifier is detected, the software on the external device can select aspecific protocol that is associated with the day of the week.

In some embodiments a patient may be provided with a plurality ofdevices to use to detect a variety of analytes. A subject may, forexample, use different devices on different days of the week. In someembodiments the software on the external device associating theidentifier with a protocol may include a process to compare the currentday with the day the device is to be used based on a clinical trial forexample. If for example, the two days of the week are not identical, theexternal device can wirelessly send notification to the subject usingany of the methods described herein or known in the art to notify themthat an incorrect device is in the system and also of the correct deviceto use that day. This example is only illustrative and can easily beextended to, for example, notifying a subject that a device is not beingused at the correct time of day.

The system can also use a networking method of assessing the medicalcondition of a subject. A system of communicating information may or maynot include a reader for reading subject data. For example, if biomarkerdata is acquired by a microfluidic point-of-care device, the valuesassigned to different individual biomarkers may be read by the deviceitself or a separate device. Another example of a reader would be a barcode system to scan in subject data that has been entered in anelectronic medical record or a physician chart. A further example of areader would consist of an electronic patient record database from whichsubject data could be directly obtained via the communications network.In this way, the efficacy of particular drugs can be demonstrated inreal-time, thus justifying reimbursement of the therapy.

Noncompliance with a medical treatment, including a clinical trial, canseriously undermine the efficacy of the treatment or trial. As such, insome embodiments the system of the present invention can be used tomonitor patient compliance and notify the patient or other medicalpersonnel of such noncompliance. For example, a patient taking apharmaceutical agent as part of medical treatment plan can take a bodilyfluid sample which is assayed as described herein, but a metaboliteconcentration, for example, detected by the system may be at an elevatedlevel compared to a known profile that will indicate multiple doses ofthe pharmaceutical agent have been taken. The patient or medicalpersonnel may be notified of such noncompliance via any or the wirelessmethods discussed herein, including without limitation notification viaa handheld device such a PDA or cell phone. Such a known profile may belocated or stored on an external device described herein.

In an embodiment, the system can be used to identify sub-populations ofpatients which are benefited or harmed by a therapy. In this way, drugswith varying toxicity that would otherwise be forced from the market canbe saved by allocating them only to those who will benefit.

Methods

The devices and methods of the invention provide an effective means forreal-time detection of analytes present in a bodily fluid from asubject. The detection methods may be used in a wide variety ofcircumstances including identification and quantification of analytesthat are associated with specific biological processes, physiologicalconditions, disorders, stages of disorders or stages of therapy. Assuch, the devices and methods have a broad spectrum of utility in, forexample, drug screening, disease diagnosis, phylogenetic classification,parental and forensic identification, disease onset and recurrence,individual response to treatment versus population bases, and monitoringof therapy. The devices and methods are also particularly useful foradvancing preclinical and clinical stage of development of therapeutics,improving patient compliance, monitoring ADRs associated with aprescribed drug, individualized medicine, outsourcing blood testing fromthe central laboratory to the residence of the patient. The device canbe employed on a prescription basis, utilized by pharmaceuticalcompanies for monitoring therapeutic agents following regulatoryapproval or utilized for payors outsourcing blood tests from a centrallab.

Accordingly, in an embodiment, the present invention provides a methodof detecting an analyte in a bodily fluid sample comprising providing ablood sample to a device or system of the invention, allowing the sampleto react within at least one assay unit of the device, and detecting thedetectable signal generated from the analyte in the blood sample.

FIG. 1 demonstrates an exemplary embodiment of a device of the inventioncomprising at least one assay unit and at least one reagent unit. Theassay units (for example, designated as sample tips and calibrator tipsin FIG. 1) can contain a capture surface and the reagent units cancontain items such as conjugates, washes, and substrates. The deviceexemplified in FIG. 1 also comprises a whole blood sample collectiontip, a plasma sample collection tip, a blood input well, a beads well orplasma separation well, a tip touch-off or blotting pad, a dilutionwell, a diluted plasma sample well or plasma diluent well, collectiontip disposal areas.

In an embodiment, a method comprises performing an Enzyme-linkedImmunosorbent Assay (ELISA). In an example as described in thisparagraph, a sample is provided to a sample collection unit of a deviceas described herein. The device is then inserted into a system, whereinsystem detects the type of cartridge or device that is inserted. Thesystem can then communicate with an external device to receive a set ofinstructions or protocol that allow the system to perform the desiredassay or assays of the cartridge. The protocol can be sent to theprogrammable processor of a fluid transfer device of the system. In anexample, the fluid transfer device engages a sample tip of the cartridgeand picks up a certain volume of the sample from the sample collectionunit and moves it to a pretreatment unit where red blood cells areremoved. The plasma of the sample can then be aspirated into a plasmatip or any assay tip by the fluid transfer device according to theprotocol. The tip containing the plasma can then pick up a diluent todilute the sample as is necessary for the assays to be run. Manydifferent dilutions can be carried by using serial dilutions of thesample. For example, each assay tip or assay unit can contain a sampleof a different dilution. After the sample is aspirated into an assayunit by the fluid transfer device, the assay unit can then be incubatedwith the sample to allow any target analyte present to attach to thecapture surface. Incubations as described in this example can be at thesystem or room temperature for any period of time, for example 10minutes, or can in a heating device of the system as described herein.The assay unit can engage a reagent unit addressed with a reagentcorresponding to the assay to be run in each individual assay unit thathave a capture surface for that assay. In this example, the firstreagent is a detector solution of an ELISA, for example, comprising adetector antibody such as a labeled anti-protein antibody different thanthe capture surface. The detector solution is then aspirated out of theassay unit and then a wash solution can be aspirated into the assay unitto remove any excess detector solution. Multiple wash steps can be used.The final reagent to be added is an enzymatic substrate which causes thebound detector solution to chemiluminesce. The enzymatic substrate isthen expelled from the assay unit and the results of the assay are readby a detector of the system. At each step as described, incubations canoccur as necessary as described herein. In this example, the entireprocess after putting the cartridge into the system is automated andcarried out by a protocol or set of instructions to the programmablesystem.

One exemplary method proceeds with delivering a blood sample into theblood input well. The sample can then be picked up by a collection tipand inserted into the plasma separation well. Alternatively, the bloodcan be deposited directly into a well containing a blood separator. Forexample, plasma separation can be carried out by a variety of methods asdescribed herein. In this example, plasma separation proceeds usingmagnetizable beads and antibodies to remove the components of the bloodthat are not plasma. The plasma can then be carried by a plasmacollection tip as to not contaminate the sample with the whole bloodcollection tip. In this example, the plasma collection tip can pick-up apredetermined amount of diluent and dilute the plasma sample. Thediluted plasma sample is then distributed to the assay units (sampletips) to bind to a capture surface. The assay units can be incubated toallow for a capture reaction to be carried out. The assay unit then canbe used to collect a conjugate to bind with the reaction in the assayunit. The conjugate can comprise an entity that allows for the detectionof an analyte of interest by a detector, such as an optical detector.Once conjugate has been added to the assay unit, the reaction can beincubated. In an exemplary method using an exemplary device of FIG. 1, areagent unit containing a wash for the conjugate is then accessed by theassay unit (sample tip) to remove any excess conjugate that caninterfere with any analyte detection. After washing away excessconjugate, a substrate can be added to the assay unit for detection. Inaddition, in the example of FIG. 1 and this method, a calibrator tipassay unit can be used to carry out all of the methods described in thisparagraph except the collection and distribution of the sample.Detection and measurements using the calibrator tip assay unit can beused to calibrate the detection and measurements of the analyte from thesample. Other processes and methods similar to those used in thisexample are described hereinafter.

Any bodily fluids suspected to contain an analyte of interest can beused in conjunction with the system or devices of the invention. Forexample, the input well or sample collection unit in the example of FIG.1 can collect of contain any type of commonly employed bodily fluidsthat include, but are not limited to blood, serum, saliva, urine,gastric and digestive fluid, tears, stool, semen, vaginal fluid,interstitial fluids derived from tumorous tissue liquids extracted fromtissue samples, and cerebrospinal fluid. In an embodiment, the bodilyfluid is blood and can be obtained by a fingerstick. In an embodiment,the bodily fluid sample is a blood plasma sample.

A bodily fluid may be drawn from a patient and distributed to the devicein a variety of ways including, but not limited to, lancing, injection,or pipetting. In one embodiment, a lancet punctures the skin anddelivers the sample into the device using, for example, gravity,capillary action, aspiration, or vacuum force. The lancet may onboardthe device, or part of a reader assembly, or a stand alone component.Where needed, the lancet may be activated by a variety of mechanical,electrical, electromechanical, or any other known activation mechanismor any combination of such methods. In another embodiment where noactive mechanism is required, a patient can simply provide a bodilyfluid to the device, as could occur, for example, with a saliva sample.The collected fluid can be placed into a collection well or unit of thedevice. In some embodiments, there is a user activated lancet and samplecollecting capillary within the device.

The volume of bodily fluid to be used with a method or device describedherein is generally less than about 500 microliters, further can bebetween about 1 to 100 microliters. Where desired, a sample of 1 to 50microliters, 1 to 40 microliters, 1 to 30 microliters, 1 to 10microliters or even 1 to 3 microliters can be used for detecting ananalyte using the subject fluidic device. In an embodiment, the sampleis 20 microliters.

In an embodiment, the volume of bodily fluid used for detecting ananalyte utilizing the devices, systems, or methods is one drop of fluid.For example, one drop of blood from a pricked finger can provide thesample of bodily fluid to be analyzed with a device, system, or methodof the invention.

In some embodiments, the bodily fluids are used directly for detectingthe analytes present in the bodily fluid without further processing.Where desired, however, the bodily fluids can be pre-treated beforeperforming the analysis with a device. The choice of pre-treatments willdepend on the type of bodily fluid used and/or the nature of the analyteunder investigation. For instance, where the analyte is present at lowlevel in a sample of bodily fluid, the sample can be concentrated viaany conventional means to enrich the analyte. Methods of concentratingan analyte include but are not limited to drying, evaporation,centrifugation, sedimentation, precipitation, and amplification. Wherethe analyte is a nucleic acid, it can be extracted using various lyticenzymes or chemical solutions or using nucleic acid binding resinsfollowing the accompanying instructions provided by manufacturers. Wherethe analyte is a molecule present on or within a cell, extraction can beperformed using lysing agents including but not limited toanticoagulants such as EDTA or heparin, denaturing detergent such as SDSor non-denaturing detergent such as Thesit, sodium deoxylate, tritonX-100, and tween-20.

In an embodiment, the subject collects a sample of bodily fluid with asyringe. The sample can enter the syringe through a capillary tube. Inan embodiment measuring an analyte in a blood sample, the subjectperforms a fingerstick and touches the outer end of the glass capillaryto the blood so that blood is drawn by capillary action and fills thecapillary with a volume. In some instances, the sample volume is known.In some embodiments, the sample volume is in the range of about 5-20microliters or other volume ranges as described herein.

In another embodiment, a method and system is provided to obtain aplasma sample substantially free of red blood cells from a blood sample.When conducting an assay, the analytes are often contained in the bloodplasma, and the red blood cells can interfere with a reaction.

Often, when measuring a blood sample, the analytes of interest are inthe serum or plasma. For clinical purposes, the final reportedconcentration of multiple blood tests often needs to relate to theconcentration of blood serum or blood plasma in a diluted sample. Inmany cases, blood serum or blood plasma is the test medium of choice inthe lab. Two operations may be necessary prior to running an assay,dilution and red blood cell removal. Blood samples vary significantly inthe proportion of the sample volume occupied by red cells (thehematocrit which varies from about 20-60%). Furthermore, in apoint-of-care environment when assay systems are operated by non-expertpersonnel, the volume of sample obtained may not be that which isintended. If a change in volume is not recognized, it can lead to errorin the reported analyte concentrations.

In related but separate embodiment, the present invention provides amethod of retrieving plasma from a blood sample is provided thatcomprises mixing a blood sample in the presence of magnetizableparticles in a sample collection unit, wherein the magnetizableparticles comprise an antibody capture surface for binding to non-plasmaportions of the blood sample, and applying a magnetic field above aplasma collection area to the mixed blood sample to effect suspension ofthe non-plasma portions of the blood sample on top of the plasmacollection area, thereby retrieving the plasma from a blood sample.

In order to process blood samples, the device or system of the inventionmay include a magnetic reagent or object which binds to red cells andenables magnetic removal of red cells from plasma. The reagent can beprovided in lyophilized form but also can be present as a liquiddispersion. A reagent comprised of magnetizable particles (for example,about 1 micrometer in size) can be coated with an antibody to a red cellantigen or to some adaptor molecule. In some embodiments, the reagentalso contains unbound antibodies to red cell surface antigens, which maybe unlabeled or labeled with an adaptor moiety (such as biotin,digoxigenin, or fluorescein). In an embodiment analyzing a blood sample,the red blood cells in a diluted sample co-agglutinate with themagnetizable particles aided by a solution phase antibody.Alternatively, a lectin that recognizes a red cell surface carbohydratecan be used as a co-agglutination agent. Sometimes, combinations of redcell agglutinating agents are used. Alternatively, a device of theinvention can comprise a blood filter, such as a pad of glass fiber, toaid in the separation of red blood cells from a sample.

When blood is mixed with a magnetic reagent, a co-agglutination canoccur in which many, if not all, of the red cells form a mixedagglutinate with the magnetizable particles. The reagent dissolution andmixing process is driven by repeated aspiration using a tip orcollection tip of the invention or a pipette-like tip. After themagnetizable mass has formed, the mass can be separated from the bloodplasma by use of a magnet to hold the mass in place as plasma is allowedto exit the tip. In an embodiment, the plasma exits the tip by gravityin a vertical orientation, while the magnet holds the mass in place. Inanother embodiment, the plasma exits the tip by vacuum or pressuremeans, while the mass is held within the tip. The plasma can bedeposited into a well, another collection tip, or assay unit of theinvention.

An example of a plasma separation method of the invention isdemonstrated in FIGS. 9A through 9E. In FIG. 9A, a whole blood sample901 has been aspirated into a sample tip 910 as described herein, forexample in the amount of about 20 microliters. The whole blood sample901 is then deposited into a separation well 920 (for example, a wellcontaining magnetic beads or particles) of an example device. FIG. 9Billustrates a method of suspending and mixing a magnetic reagent in thewhole blood sample 902 in a separation well (for example, magnetic beadparticles and free binding molecules). FIG. 9C demonstrates a 10microliter air slug 930 that can be used to prevent loss from the tip910. The mixed whole blood sample and magnetic reagent 902 are incubatedfor several seconds (for example, 60 to 180 seconds) to allow anagglutination reaction to occur.

FIG. 9D demonstrates the application of a magnetic field 940 to thewhole blood cell and magnetic reagent mixture 902. The magnetic field940 can be applied by a magnetic collar 942 that is incorporated with asystem or with any magnetic means known in the art. The magnetic field940 attracts any particles that have adhered to the magnetic reagent. Inthis way, the plasma 903, which does not adhere with the magneticreagent, can be separated from non-plasma portions of a whole bloodsample.

FIG. 9E demonstrates a method of distributing a blood plasma sample 903,as separated by the magnetic reagent described herein, into a well orunit 950 of a device as described herein. The blood plasma sample 903can also be distributed to a collection tip or assay unit, as well asany other sort of assay device as obvious to one skilled in the art. InFIG. 9E, the magnetic field 940 is shown to move with the tip 910distributing the blood plasma sample 903. In this example, 5 to 8microliters of plasma have been removed from a 20 microliter whole bloodsample. 1 to 99% of a whole blood sample can be plasma separated using amethod of the invention. In an embodiment, 25 to 60% of the volume ofthe whole blood sample is plasma that can be separated.

Other exemplary steps of a method as described can be completed. Inorder to move the blood plasma sample to another well or unit, acapillary plasma collection tip (which can be operated by a roboticsystem or any other system of the invention) collects the blood plasmasample by capillary and aspiration force. Another step can comprisedistributing the plasma sample in a diluent, and the sample can then bediluted by the diluent. The diluted blood plasma sample can then becollected by the collection tip in a predetermined volume. The dilutedblood plasma sample can then be mixed and distributed into a well orunit of a device to be distributed to one or a plurality of assay unitsof a device of the invention. The sample can also be distributed intoany other type of device, such as a microtiter plate, as would beobvious to those skilled in the art.

The example process demonstrated in FIGS. 9A through 9E can be used withother devices and systems, other than those disclosed herein. Forexample, a fluid transfer tip can contain the agglutinated mass and theplasma could be deposited into a microtiter plate. Other devices andsystems as would be obvious to those skilled in the art could beutilized to execute the example blood plasma separation as disclosedherein.

The sample of bodily fluid can also be diluted in a variety of othermanners, such as using a sample collection device capable of dilution.The housing of the sample collection device can comprise a tube. In thetube, two moveable seals can contain a volume of a diluent. In apreferable embodiment, the volume of the diluent is predetermined, e.g.,in about the range of 50 microliters to 1 milliliter, preferably in therange of about 100 microliters to 500 microliters.

In an aspect, a method for automated detection of a plurality ofanalytes in a bodily fluid sample is provided that comprises: providingthe bodily fluid sample to a fluidic device, wherein the fluidic devicecomprises: a sample collection unit configured to contain the bodilyfluid sample; an array of assay units, wherein an individual assay unitof said array of assay units is configured to run a chemical reactionthat yields a signal indicative of an individual analyte of saidplurality of analytes being detected; and an array of reagent units,wherein an individual reagent unit of said array of reagent unitscontains a reagent. The method can also comprise engaging the individualassay unit using a fluid transfer device. Continuing the method, bodilyfluid sample can be transferred from the sample collection unit to theindividual assay unit using the fluid transfer device and the reagentfrom the individual reagent unit can be transferred to the individualassay unit, thereby reacting the reagent with the bodily fluid sample toyield the signal indicative of the individual analyte of the pluralityof analytes being detected. In some embodiments, the fluid transferdevice comprises a plurality of heads, wherein an individual head of theplurality of heads is configured to engage the individual assay unit;and wherein said fluid transfer device comprises a programmableprocessor configured to direct fluid transfer of the bodily fluid samplefrom the sample collection unit and the reagent from the individualreagent unit into the individual assay unit.

In some instances, instructions are provided to the programmableprocessor, for example, by a user, a subject, or the manufacturer.Instructions can be provided from an external device, such as a personalelectronic device or a server. The instructions can direct the step oftransferring the bodily fluid sample to the individual assay unit. Forexample, the step of transferring the bodily fluid sample can affect adegree of dilution of the bodily fluid sample in the individual assayunit to bring the signal indicative the individual analyte of theplurality of analytes being detected within a detectable range. In someexamples, the degree of dilution of the bodily fluid sample brings thesignals indicative of the at least two individual analytes within adetectable range as described herein.

Pattern recognition techniques can be used to determine if the detectionof an analyte or a plurality of analytes by a method as described hereinis within or outside a certain range. For example, detectable signalsoutside the reportable range can be rejected. The certain range can beestablished during calibration of a fluidic device the reagent and assayunits. For example, the range is established when a device is assembledin a just-in-time fashion.

In some instances, if the detectable signal of an analyte as detectedwith a lower dilution factor or degree of dilution exceeds that for ahigher dilution factor, the lower dilution result can be rejected asinvalid. In most instances, concentrations of an analyte in a sample asderived from signals from samples with different degrees of dilution getlower as the degree of dilution becomes greater. If this does happen, anassay result can be verified. The systems, devices, and methods hereinprovide the flexibility of quality control rules such as those describedthat many POC devices cannot offer. The systems, devices, and methodsprovide many of the quality control features as would be expected in alaboratory setting.

In an embodiment, a sample is diluted in a ratio that is satisfactoryfor both high senstivity and low sensitivity assays. For example, adilution ratio of sample to diluent can be in the range of about1:10,000-1:1. The device can enable a sample to be diluted into separatelocations or extents. The device can also enable the sample to besubject to serial dilutions. In further instances, serial dilutionwithin the device or system can dilute the sample up to10,000,000,000:1.

In embodiments, a sample containing an analyte for detection can bemoved from a first location to a second location by aspiration-,syringe-, or pipette-type action. The sample can be drawn into thereaction tip by capillary action or reduced atmospheric pressure. Insome embodiments, the sample is moved to many locations, including anarray of assay units of a device of the invention and different wells inthe housing of a device of the invention. The process of moving thesample can be automated by a system of the invention, as describedherein.

The assay units and/or collection tips containing the sample can also bemoved from a first location to a second location. The process of movingan assay unit or a collection tip can be automated and carried out by auser-defined protocol.

In an embodiment, the assay units are moved to collect reagent from areagent unit of the invention. In many embodiments, movement of an assayunit is automated. Aspiration-, syringe-, or pipette-type action can beused to collect reagent from a reagent unit into an assay unit.

Once a sample has been added to an assay unit that comprises a capturesurface, the entire unit can be incubated for a period of time to allowfor a reaction between the sample and the capture surface of the assayunit. The amount of time needed to incubate the reaction is oftendependent on the type of assay being run. The process can be automatedby a system of the invention. In an embodiment, the incubation time isbetween 30 seconds and 60 minutes. In another embodiment, the incubationtime is 10 minutes.

An assay unit can also be incubated at an elevated temperature. In anembodiment, the assay unit is incubated at temperature in a range ofabout 20 to 70 degrees Celsius. The assay unit can be inserted into aheating block to elevate the temperature of the assay unit and/or thecontents of the assay unit.

In an embodiment of a method of the invention, a conjugate is added tothe assay unit after a sample has been added to the unit. The conjugatecan contain a molecule for labeling an analyte captured by a capturesurface in the assay unit. Examples of conjugates and capture surfaceare described hereinafter. The conjugate can be a reagent containedwithin a reagent unit. The conjugate can be distributed to the assayunit by aspiration-, syringe-, or pipette-type action. Once a conjugatehas been distributed to an assay unit, the assay unit can be incubatedto allow the conjugate to react with an analyte within the assay unit.The incubation time can be determined by the type of assay or theanalyte to be detected. The incubation temperature can be anytemperature appropriate for the reaction.

In an aspect, a method of calibrating a device for automated detectionof an analyte in a bodily fluid sample is provided. A device cancomprise an array of addressable assay units configured to run achemical reaction that yields a detectable signal indicative of thepresence or absence of the analyte, and an array of addressable reagentunits, each of which is addressed to correspond to one or moreaddressable assay units in said device, such that individual reagentunits are calibrated in reference to the corresponding assay unit(s)before the arrays are assembled on the device. The device is calibratedby calibrating the assay units and reagent units before they areassembled on the device. The device can then be assembled using thecalibrated components, making the device, and a method and system thatutilize the device, modular components.

Calibration can be pre-established by measuring the performance of assayreagents, such as conjugates, before the assay units and reagent unitare assembled in a device of the invention. Calibration information andalgorithms can be stored on a server linked wirelessly to the assaysystem. Calibration can be performed in advance or retrospectively byassays performed in replicate systems at a separate location or by usinginformation obtained when the assay system is used.

In an aspect, a control material can be used in a device or system tomeasure or verify the extent of dilution of a bodily fluid sample. Forexample, another issue of solid-phase based assays such as ELISA is thatan assay uses a solid-phase reagent that is difficult to quality controlwithout destruction of its function. The systems and methods hereinprovide methods to determine the dilution achieved in a POC system usinga disposable device with automated mixing and/or dilution.

In an embodiment, a method provides retrospective analysis, for example,by use of a server in real time to analyze data prior to reportingresults. For example, an assay can be performed and a control assay canbe run in parallel to the assay. The control assay provides ameasurement of an expected dilution of the sample. In some examples, thecontrol assay can verify the dilution of the sample and thus, dilutionof a sample for the assay or plurality of assays run within the systemcan be considered accurate.

A method of measuring a volume of a liquid sample can comprise: reactinga known quantity of a control analyte in a liquid sample with a reagentto yield a detectable signal indicative of the control analyte; andcomparing an intensity of said detectable signal with an expectedintensity of said detectable signal, wherein the expected intensity ofsaid signal is indicative of an expected volume of the liquid sample,and wherein said comparison provides a measurement of said volume ofsaid liquid sample being measured. In many instances, the controlanalyte is not present in said liquid sample in a detectable amount.

In an embodiment, a method can further comprise verifying the volume ofsaid liquid sample when the measurement of the volume of the sample iswithin about 50% of the expected volume of the liquid sample.

For example, a method utilized a device or system described herein canfurther comprise: reacting a bodily fluid sample containing a targetanalyte with a reagent to yield a detectable signal indicative of thetarget analyte; and measuring the quantity of the target analyte in thebodily fluid sample using an intensity of said detectable signalindicative of the target analyte and the measurement of said volume ofsaid liquid sample. The liquid sample and the bodily fluid sample can bethe same sample. In some embodiments, the control analyte does not reactwith the target analyte in the bodily fluid sample, therefore providingnot interacting with detection of the target analyte.

In some instances, the liquid sample and the bodily fluid sample aredifferent liquid samples. For example, a control liquid, such as water,and a blood sample. Or in another example, a saliva sample and a bloodsample.

A control analyte can be, without limitation, fluorescein-labeledalbumin, fluorescein labeled IgG, anti-fluorescein, anti-digoxigenin,digoxigenin-labeled albumin, digoxigenin-labeled IgG, biotinylatedproteins, non-human IgG. Other exemplary control analytes can be obviousto one skilled in the art. In an embodiment, the control analyte doesnot occur in a human bodily fluid sample.

In a POC system as described herein configured to detect a plurality ofanalytes within a sample, the system can have capabilities to dilute andmix liquids. In many instances, an automated system or user can use acontrol assay to measure the dilution actually achieved and factor thatdilution into the system calibration. For example, a control analyte canbe never found in the sample of interest and dried into a reagent unit.The quantity of the dried control analyte can be known and mixed with asample in the reagent unit. The concentration of analyte can be measuredto indicate the volume of sample and any dilution performed on thesample.

Examples of control analytes for an immunoassay include, but are notlimited to: fluorescein-labeled protein, biotinylated protein,fluorescein-labeled, Axlexa™-labeled, Rhodamine-labeled, TexasRed-labeled, immunoglobulin. For example the labelingcan be achieved byhaving at least two haptens linked per molecule of protein. In someembodiments, 1-20 haptens are linked per molecule of protein. In afurther embodiment, 4-10 haptens are linked per molecule of protein.Many proteins have large numbers of free amino groups to which thehaptens can be attached. In many instances, hapten-modified proteins arestable and soluble. Also, haptens such as fluorescein and Texas Red aresufficiently large and rigid that antibodies with high affinity can bemade (for example, a hapten is large enough to fill the antibody bindingsite). In some embodiments, haptens can be attached to proteins usingreagents, such as fluorescein isothocyanate, and fluorescein carboxylicacid NHS ester to create control analytes in which the part recognizedby the assay system is the hapten.

In some embodiments, a method utilizes dried control analyte. In someexamples, a dried control analyte avoids dilution of the sample and canmake the control analyte more stable. Dried control analyte can beformulated so it dissolves rapidly and/or completely on exposure to aliquid sample. In some embodiments, a control analyte can be an analytefor which antibodies with high affinity. In some instances, a controlanalyte can be an analyte that has no cross reaction with any endogenoussample component. Additionally, for example, the analyte can beinexpensive and/or easy to make. In some embodiments, the controlanalyte is stable over the lifetime of the device or system describedherein. Exemplary carriers used to create analytes with covalentlylinked haptens include proteins such as, but not limited to: albumin,IgG, and casein. Exemplary polymer carriers used to create novelanalytes with covalently linked haptens include, but are not limited to:Dextran, Poly-vinylpyrolidone. Exemplary excipients used to formulateand stabilize control analytes include, but are not limited to: sucrose,salts, and buffers (such as sodium phosphate and tris chloride).

A control analyte and method as described herein can be used in avariety of ways including the examples described herein. For example, amethod can measure a volume of a sample. In some embodiments, a methodmeasures dilution or a dilution factor or a degree of dilution of asample. In some instances, a method provides a concentration of thecontrol analyte in a sample. In a system or device described herein todetect a plurality of analytes, measurements from a method herein usinga control analyte can be used to verify or describe measurements oftarget analytes. For example, a fluid transfer device with multipleheads may be used to distribute liquid into a plurality of assay units,including a control unit. In some instances, it can be assumed thatliquid amount distributed into the plurality of units is the same orsimilar between the individual units. In some embodiments, a methoddescribed herein with a control analyte can be used to verify that thecorrect volume of sample has been collected or utilized within a deviceor system. In another embodiment, a method verifies the correct volumeof diluent has been provided to the sample. Also, the dilution factor ordegree of dilution can also be verified. In yet another embodiment, amethod with a control analyte verifies the correct volume of dilutedsample has been distributed to the plurality of units.

FIG. 10 demonstrates an exemplary method of a control assay as describedherein comprising a known quantity of control analyte. A unit 1010before assembly into a cartridge can be filled with a solution 1001comprising a known mass of control analyte 1002. The liquid of thesolution can be removed and the unit 1010 dried to leave the controlanalyte 1002 in the unit 1010. The unit 1010 can then be inserted into adevice and transported for use. When the unit 1010 is used and receivesa sample or diluent 1003, the sample 1003 can be delivered in anexpected volume and mixed with the dried control analyte 1002 within theunit 1010 to create a control solution 1004 with an expectedconcentration. The control solution 1004 can be optionally diluted. Inan embodiment, the control analyte 1002 can be detected by the samemanners as a target analyte in the device. The control analyteconcentration in the control solution 1004 is measured. The measurementof the concentration can be used to calculate the volume of the sample1003 added to create the control solution 1004. In this manner, a usercan compare the measured volume of the sample 1003 with the expectedvolume of the sample 1003.

In an example, red blood cells can be removed from a blood sample.However, if some red blood cells remain, or red blood cells are notremoved from a blood sample, a method with a control analyte can be usedto correct for effects from red blood cells in the blood sample. Becausehematocrit can vary significantly (for example, from 20-60% of the totalvolume of a sample), the quantity of an analyte in a fixed or expectedvolume (v) of blood can be a function of the hematocrit (H expressedhere as a decimal fraction). For example, the quantity of analyte with aconcentration C in plasma is C*v*(1-H). Thus the quantity for a samplewith hematocrit 0.3 is 1.4 times that for a sample with hematocrit 0.5.In an exemplary embodiment, undiluted blood can be dispensed into adevice as described and red cells can be removed. A control analyteconcentration in the plasma fraction can then be measured to estimatethe volume of sample plasma and determine the hematocrit.

In some embodiments, unbound conjugate may need to be washed from areaction site to prevent unbound conjugates from producing inaccuratedetection. The limiting step of many immunoassays is a washing step. Thecompromise of minimum carryover and high sensitivity is dependent on thewash removal of unbound conjugate. The wash step can be severely limitedin a microtiter plate format due to the difficulty of removing the washliquid from a well (for example, by automatic means). An assay unitdevice and system of the invention can have a number of advantages inthe way liquids are handled. An advantage may be an improvement in thesignal to noise ratio of an assay.

Removal of the conjugate can be difficult to if conjugates are stickingto the edges of the assay units of a device if, for example, there isnot an excess of a wash solution.

A wash of the conjugate can occur by either pushing the wash solutionfrom above or drawing the wash solution up and expelling the liquidsimilar to the loading of the sample. The washing can be repeated asmany times as necessary.

When using a wash buffer in an assay, the device can store the washbuffer in reagent units and the assay unit can be brought into fluidcommunication with the wash. In an embodiment, the wash reagent is ableto remove unbound reagent from the assay units by about 99, 99.9, or99.999% by washing. In general, a high washing efficiency resulting in ahigh degree of reduction of undesired background signals is preferred.Washing efficiency is typically defined by the ratio of signal from agiven assay to the total amount of signal generated by an assay with nowash step and can be readily determined by routine experimentation. Itcan be generally preferred to increase the volume of washing solutionand time of incubation but without sacrificing the signals from a givenassay. In some embodiments, washing is performed with about 50 ul toabout 5000 ul of washing buffer, preferably between about 50 ul to about500 ul washing buffer, for about 10 to about 300 seconds.

Additionally, it can be advantageous to use several cycles of smallvolumes of wash solution which are separated by periods of time where nowash solution is used. This sequence allows for diffusive washing, wherelabeled antibodies diffuse over time into the bulk wash solution fromprotected parts of the assay unit such as the edges or surfaces where itis loosely bound and can then be removed when the wash solution is movedfrom the reaction site.

In many embodiments, the last step is to distribute an enzymaticsubstrate to detect the conjugate by optical or electrical means.Examples of substrates are described hereinafter.

For example, the reagent in the individual reagent unit of a deviceherein can be an enzyme substrate for an immunoassay. In anotherembodiment, the step of transferring the substrate reagent from theindividual reagent unit can be repeated after a reaction at the capturesite. For example, enzymatic substrate is transferred to a reaction siteand incubated. After measuring the assay signal produced, used substratecan be removed and replaced with fresh substrate and the assay signalremeasured. A signal indicative of the individual analyte being can bedetected using a system as described herein from both the first and thesecond application of substrate. The second substrate is usually thesame as the original substrate. In an embodiment, the second substrateis transferred to a reaction site from a second reagent unit of a deviceherein. In another embodiment, the second substrate is transferred to areaction site from the same reagent unit as the original substrate.Transferring a second substrate thereby creates a second reaction toyield a second signal indicative of the individual analyte. Theintensity of the original signal and a second intensity of the secondsignal can be compared to calculate the final intensity of the signalindicative of the individual analyte and whether the assay was properlyconducted.

In an embodiment, the intensities of the multiple signals can be usedfor quality control of an assay. For example, if the signals differ by20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, the assay resultsmay be disregarded.

In an embodiment, a method as described herein comprises re-loadingsample and or detector-conjugate (enzyme-labeled antibody) and or theenzyme substrate and sample to rectify or confirm an assay signal or touse as an internal control. For example, re-use of an assay tip or unitas described can be provided to verify function and/or to add furthersample or control materials obtain a second signal.

In some instances, a method of re-loading a substrate to an enzyme unitis enabled by the ability of a system as described herein toautomatically to transfer liquid samples and reagents into the assayunits. Some assays do not require the system to deliver a resultimmediately or on a schedule, therefore, a control method as describedoffers an opportunity to possibly enhance the reliability of theresults. A response observed following iterations of adding an enzymesubstrate can be used to verify the initial response or to calculatespike recovery.

Experiments have shown that by adding a second enzyme substrate to anassay unit, the reproducibility of results can be maintained. In someembodiments, a control method provides replicate analyses using an assayunit gave a response significantly lower than that expected.

With any control methods described herein, there are numerous possibleerrors that can be accounted for or postulated from executing a controlmethod. Exemplary assay errors include, but are not limited to, impropermanufacturing of an assay unit or device, improper aspiration of asample and/or one or more reagents, an assay unit is not positionedproperly relative to the photomultiplier during detection, and a missingassay unit in the device or system.

In some embodiments, the present invention provides a method ofobtaining pharmacological data useful for assessing efficacy and/ortoxicity of a pharmaceutical agent from a test animal utilizing thesubject fluidic devices or systems.

When using laboratory animals in preclinical testing of a pharmaceuticalagent, it is often necessary to kill the test subject to extract enoughblood to perform an assay to detect an analyte of interest. This hasboth financial and ethical implications, and as such it may beadvantageous to be able to draw an amount of blood from a test animalsuch that the animal does not need to be killed. In addition, this canalso allow the same test animal to be tested over several different timepoints, thus allowing for a more effective evaluation of the effects ofan agent on single animals. On average, the total blood volume in amouse, for example, is 6-8 mL of blood per 100 gram of body weight. Abenefit of the current invention is that only a very small volume ofblood is required to perform preclinical trials on mice or other smalllaboratory animals. In some embodiments between about 1 microliter andabout 50 microliters are drawn. In an embodiment between about 1microliter and 10 microliters are drawn. In preferred embodiments about5 microliters of blood are drawn.

A further advantage of keeping the test animal alive is evident in apreclinical time course study. When multiple mice, for example, are usedto monitor the levels of an analyte in a test subject's bodily fluidover time, the added variable of using multiple subjects is introducedinto the trial. When, however, a single test animal can be used as itsown control over a course of time, a more accurate and beneficialpreclinical trial can be performed.

In some embodiments a method of automatically monitoring patientcompliance with a medical treatment using the subject devices or systemsis provided. The method comprises the steps of allowing a sample ofbodily fluid to react with assay reagents in a device to yield adetectable signal indicative of the presence of an analyte in saidsample; detecting said signal with said device; comparing said signalwith a known profile associated with said medical treatment to determineif said patient is compliant or noncompliant with said medicaltreatment; and notifying a patient of said compliance or noncompliance.

In another embodiment, the system and methods of the invention provide ameans of discovering new biomarkers and/or validating by association oftrends in such markers with disease and therapy outcomes.

In another embodiment, the system and methods of the invention canidentify trends in biomarker levels and daily patient diary informationover time that can be used to adjust a drug dose to an optimal level forparticular patients (for example, adaptive dose-ranging).

In some embodiments noncompliance may include taking an improper dose ofa pharmaceutical agent including without limitation multiple doses or nodose, or may include inappropriately mixing pharmaceutical agents. Inpreferred embodiments a patient is notified substantially immediatelyafter the signal is compared with a known profile.

A patient or subject of a clinical trial may forget to take a bodilyfluid sample for analysis as described herein. In some embodiments amethod of alerting a patient to test a sample of bodily fluid using adevice as described herein comprises providing a protocol to be run onsaid device, said protocol located on an external device, associatedwith said patient, and comprising a time and date to test said sample ofbodily fluid; and notifying patient to test said bodily fluid on saiddate and time if said sample has not been tested. In some embodiments apatient can be notified wirelessly as described herein. Compliance withtherapeutic regimes can be improved by use of prompts on a display andobtaining responses from patients (for example, by way of atouch-screen).

A patient may be provided with a device when procuring a prescription ofdrugs by any common methods, for example, at a pharmacy. Likewise, aclinical trial subject may be provided with such devices when starting aclinical trial. The patient or subject's contact information, includingwithout limitation cell phone, email address, text messaging address, orother means of wireless communication, may at that time be entered intothe external device and associated with the patient or subject asdescribed herein, for example, in a database. Software on the externaldevice may include a script or other program that can detect when asignal generated from a detection device has not yet been sent to theexternal device, for example at a given time, and the external devicecan then send an alert notifying the patient to take a bodily fluidsample.

In one embodiment, the system is provided directly to a consumer and isused in lifestyle and/or athletic management. Relevant lifestyle andexercise data can be input and measurements of parameters indicative ofmuscle damage, anaerobic metabolism (for example, lactic acid) can bemeasured. In some embodiments, the system can be sufficiently small tobe portable.

In another embodiment, the system is particularly suited for measurementof markers in the blood of small animals such as rats and mice that arecommonly used in pre-clinical work. Such animals only have a smallvolume of blood and so assay systems requiring very small volumes ofsample are particularly useful, especially in longitudinal studies whereseveral samples from a single animal are needed in rapid succession.These considerations can be especially important when several analytesneed to be measured in parallel.

In one embodiment, the system includes a convenient way to package theseveral elements required for multiple complex assays in a secure formfor shipping. For example, assay elements click fit into a housing.

Assays

A variety of assays may be performed on a fluidic device according tothe present invention to detect an analyte of interest in a sample. Awide diversity of labels is available in the art that can be employedfor conducting the subject assays. In some embodiments labels aredetectable by spectroscopic, photochemical, biochemical,electrochemical, immunochemical, or other chemical means. For example,useful nucleic acid labels include the radioisotopes 32P, 35S,fluorescent dyes, electron-dense reagents, enzymes. A wide variety oflabels suitable for labeling biological components are known and arereported extensively in both the scientific and patent literature, andare generally applicable to the present invention for the labeling ofbiological components. Suitable labels include radionucleotides,enzymes, substrates, cofactors, inhibitors, fluorescent moieties,chemiluminescent moieties, bioluminescent labels, or colorimetriclabels. Reagents defining assay specificity optionally include, forexample, monoclonal antibodies, polyclonal antibodies, proteins, nucleicacid probes or other polymers such as affinity matrices, carbohydratesor lipids. Detection can proceed by any of a variety of known methods,including spectrophotometric or optical tracking of radioactive,fluorescent, or luminescent markers, or other methods which track amolecule based upon size, charge or affinity. A detectable moiety can beof any material having a detectable physical or chemical property. Suchdetectable labels have been well-developed in the field of gelelectrophoresis, column chromatography, solid substrates, spectroscopictechniques, and the like, and in general, labels useful in such methodscan be applied to the present invention. Thus, a label includes withoutlimitation any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, nucleic acid probe-based, electrical,optical thermal, or other chemical means.

In some embodiments the label is coupled directly or indirectly to amolecule to be detected such as a product, substrate, or enzyme,according to methods well known in the art. As indicated above, a widevariety of labels are used, with the choice of label depending on thesensitivity required, ease of conjugation of the compound, stabilityrequirements, available instrumentation, and disposal provisions.Non-radioactive labels are often attached by indirect means. Generally,a receptor specific to the analyte is linked to a signal generatingmoiety. Sometimes the analyte receptor is linked to an adaptor molecule(such as biotin or avidin) and the assay reagent set includes a bindingmoiety (such as a biotinylated reagent or avidin) that binds to theadaptor and to the analyte. The analyte binds to a specific receptor onthe reaction site. A labeled reagent can form a sandwich complex inwhich the analyte is in the center. The reagent can also compete withthe analyte for receptors on the reaction site or bind to vacantreceptors on the reaction site not occupied by analyte. The label iseither inherently detectable or bound to a signal system, such as adetectable enzyme, a fluorescent compound, a chemiluminescent compound,or a chemiluminogenic entity such as an enzyme with a luminogenicsubstrate. A number of ligands and anti-ligands can be used. Where aligand has a natural anti-ligand, for example, biotin, thyroxine,digoxigenin, and cortisol, it can be used in conjunction with labeled,anti-ligands. Alternatively, any haptenic or antigenic compound can beused in combination with an antibody.

In some embodiments the label can also be conjugated directly to signalgenerating compounds, for example, by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansylgroups, and umbelliferone. Chemiluminescent compounds includedioxetanes, acridinium esters, luciferin, and2,3-dihydrophthalazinediones, such as luminol.

Methods of detecting labels are well known to those of skilled in theart. Thus, for example, where the label is radioactive, means fordetection include scintillation counting or photographic films as inautoradiography. Where the label is fluorescent, it may be detected byexciting the fluorochrome with light of an appropriate wavelength anddetecting the resulting fluorescence by, for example, microscopy, visualinspection, via photographic film, by the use of electronic detectorssuch as digital cameras, charge coupled devices (CCDs) orphotomultipliers and phototubes, or other detection device. Similarly,enzymatic labels are detected by providing appropriate substrates forthe enzyme and detecting the resulting reaction product. Finally, simplecolorimetric labels are often detected simply by observing the colorassociated with the label. For example, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

In some embodiments the detectable signal may be provided byluminescence sources Luminescence is the term commonly used to refer tothe emission of light from a substance for any reason other than a risein its temperature. In general, atoms or molecules emit photons ofelectromagnetic energy (e.g., light) when then move from an excitedstate to a lower energy state (usually the ground state). If excitingcause is a photon, the luminescence process is referred to asphotoluminescence. If the exciting cause is an electron, theluminescence process can be referred to as electroluminescence. Morespecifically, electroluminescence results from the direct injection andremoval of electrons to form an electron-hole pair, and subsequentrecombination of the electron-hole pair to emit a photon. Luminescencewhich results from a chemical reaction is usually referred to aschemiluminescence. Luminescence produced by a living organism is usuallyreferred to as bioluminescence. If photoluminescence is the result of aspin-allowed transition (e.g., a single-singlet transition,triplet-triplet transition), the photoluminescence process is usuallyreferred to as fluorescence. Typically, fluorescence emissions do notpersist after the exciting cause is removed as a result of short-livedexcited states which may rapidly relax through such spin-allowedtransitions. If photoluminescence is the result of a spin-forbiddentransition (e.g., a triplet-singlet transition), the photoluminescenceprocess is usually referred to as phosphorescence. Typically,phosphorescence emissions persist long after the exciting cause isremoved as a result of long-lived excited states which may relax onlythrough such spin-forbidden transitions. A luminescent label may haveany one of the above-described properties.

Suitable chemiluminescent sources include a compound which becomeselectronically excited by a chemical reaction and may then emit lightwhich serves as the detectible signal or donates energy to a fluorescentacceptor. A diverse number of families of compounds have been found toprovide chemiluminescence under a variety or conditions. One family ofcompounds is 2,3-dihydro-1,4-phthalazinedione. A frequently usedcompound is luminol, which is a 5-amino compound. Other members of thefamily include the 5-amino-6,7,8-trimethoxy- and thedimethylamino[ca]benz analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino and -methoxy substituents.Chemiluminescence may also be obtained with oxalates, usually oxalylactive esters, for example, p-nitrophenyl and a peroxide such ashydrogen peroxide, under basic conditions. Other useful chemiluminescentcompounds that are also known include —N-alkyl acridinum esters anddioxetanes. Alternatively, luciferins may be used in conjunction withluciferase or lucigenins to provide bioluminescence.

The term analytes as used herein includes without limitation drugs,prodrugs, pharmaceutical agents, drug metabolites, biomarkers such asexpressed proteins and cell markers, antibodies, serum proteins,cholesterol and other metabolites, polysaccharides, nucleic acids,biological analytes, biomarkers, genes, proteins, or hormones, or anycombination thereof. Analytes can be combinations of polypeptides,glycoproteins, polysaccharides, lipids, and nucleic acids.

Of particular interest are biomarkers are associated with a particulardisease or with a specific disease stage. Such analytes include but arenot limited to those associated with autoimmune diseases, obesity,hypertension, diabetes, neuronal and/or muscular degenerative diseases,cardiac diseases, endocrine disorders, metabolic disorders,inflammation, cardiovascular diseases, sepsis, angiogenesis, cancers,Alzheimer's disease, athletic complications, and any combinationsthereof.

Of also interest are biomarkers that are present in varying abundance inone or more of the body tissues including heart, liver, prostate, lung,kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, andselected tissues that are affected by various disease, such as differenttypes of cancer (malignant or non-metastatic), autoimmune diseases,inflammatory or degenerative diseases.

Also of interest are analytes that are indicative of a microorganism,virus, or Chlamydiaceae. Exemplary microorganisms include but are notlimited to bacteria, viruses, fungi and protozoa. Analytes that can bedetected by the subject method also include blood-born pathogensselected from a non-limiting group that consists of Staphylococcusepidermidis, Escherichia coli, methicillin-resistant Staphylococcusaureus (MSRA), Staphylococcus aureus, Staphylococcus hominis,Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus capitis,Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus influenzae,Staphylococcus simulans, Streptococcus pneumoniae and Candida albicans.

Analytes that can be detected by the subject method also encompass avariety of sexually transmitted diseases selected from the following:gonorrhea (Neisseria gorrhoeae), syphilis (Treponena pallidum), clamydia(Clamyda tracomitis), nongonococcal urethritis (Ureaplasm urealyticum),yeast infection (Candida albicans), chancroid (Haemophilus ducreyi),trichomoniasis (Trichomonas vaginalis), genital herpes (HSV type I &II), HW I, HIV II and hepatitis A, B, C, G, as well as hepatitis causedby TTV.

Additional analytes that can be detected by the subject methodsencompass a diversity of respiratory pathogens including but not limitedto Pseudomonas aeruginosa, methicillin-resistant Staphlococccus aureus(MSRA), Klebsiella pneumoniae, Haemophilis influenzae, Staphylococcusaureus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae,Escherichia coli, Enterococcus faecalis, Serratia marcescens,Haemophilis parahaemolyticus, Enterococcus cloacae, Candida albicans,Moraxiella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii,Enterococcus faecium, Klebsella oxytoca, Pseudomonas fluorscens,Neiseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii,Klebsella pneumoniae Legionella pneumophila, Mycoplasma pneumoniae, andMycobacterium tuberculosis.

Listed below are additional exemplary markers according to the presentinvention: Theophylline, CRP, CKMB, PSA, Myoglobin, CA125, Progesterone,TxB2, 6-keto-PGF-1-alpha, and Theophylline, Estradiol, Lutenizinghormone, Triglycerides, Tryptase, Low density lipoprotein Cholesterol,High density lipoprotein Cholesterol, Cholesterol, IGFR.

Exemplary liver markers include without limitation LDH, (LD5), (ALT),Arginase 1 (liver type), Alpha-fetoprotein (AFP), Alkaline phosphatase,Alanine aminotransferase, Lactate dehydrogenase, and Bilirubin.

Exemplary kidney markers include without limitation TNFa Receptor,Cystatin C, Lipocalin-type urinary prostaglandin D, synthatase (LPGDS),Hepatocyte growth factor receptor, Polycystin 2, Polycystin 1,Fibrocystin, Uromodulin, Alanine, aminopeptidase,N-acetyl-B-D-glucosaminidase, Albumin, and Retinol-binding protein(RBP).

Exemplary heart markers include without limitation Troponin I (TnI),Troponin T (TnT), CK, CKMB, Myoglobin, Fatty acid binding protein(FABP), CRP, D-dimer, S-100 protein, BNP, NT-proBNP, PAPP-A,Myeloperoxidase (MPO), Glycogen phosphorylase isoenzyme BB (GPBB),Thrombin Activatable Fibrinolysis Inhibitor (TAFI), Fibrinogen, Ischemiamodified albumin (IMA), Cardiotrophin-1, and MLC-I (Myosin LightChain-I).

Exemplary pancrease markers include without limitation Amylase,Pancreatitis-Assocoated protein (PAP-1), and Regeneratein proteins(REG).

Exemplary muscle tissue markers include without limitation Myostatin.

Exemplary blood markers include without limitation Erythopoeitin (EPO).

Exemplary bone markers include without limitation, Cross-linkedN-telopeptides of bone type I collagen (NTx) Carboxyterminalcross-linking telopeptide of bone collagen, Lysyl-pyridinoline(deoxypyridinoline), Pyridinoline, Tartrate-resistant acid phosphatase,Procollagen type I C propeptide, Procollagen type I N propeptide,Osteocalcin (bone gla-protein), Alkaline phosphatase, Cathepsin K, COMP(Cartillage Oligimeric Matrix Protein), Osteocrin Osteoprotegerin (OPG),RANKL, sRANK, TRAP 5 (TRACP 5), Osteoblast Specific Factor 1 (OSF-1,Pleiotrophin), Soluble cell adhesion molecules, sTfR, sCD4, sCD8, sCD44,and Osteoblast Specific Factor 2 (OSF-2, Periostin).

In some embodiments markers according to the present invention aredisease specific. Exemplary cancer markers include without limitationPSA (total prostate specific antigen), Creatinine, Prostatic acidphosphatase, PSA complexes, Prostrate-specific gene-1, CA 12-5,Carcinoembryonic Antigen (CEA), Alpha feto protein (AFP), hCG (Humanchorionic gonadotropin), Inhibin, CAA Ovarian C1824, CA 27.29, CA 15-3,CAA Breast C1924, Her-2, Pancreatic, CA 19-9, Carcinoembryonic Antigen,CAA pancreatic, Neuron-specific enolase, Angiostatin DcR3 (Soluble decoyreceptor 3), Endostatin, Ep-CAM (MK-1), Free Immunoglobulin Light ChainKappa, Free Immunoglobulin Light Chain Lambda, Herstatin, ChromograninA, Adrenomedullin, Integrin, Epidermal growth factor receptor, Epidermalgrowth factor receptor-Tyrosine kinase, Pro-adrenomedullin N-terminal 20peptide, Vascular endothelial growth factor, Vascular endothelial growthfactor receptor, Stem cell factor receptor, c-kit/KDR, KDR, and Midkine.

Exemplary infectious disease conditions include without limitation:Viremia, Bacteremia, Sepsis, and markers: PMN Elastase, PMNelastase/α1-PI complex, Surfactant Protein D (SP-D), HBVc antigen, HBVsantigen, Anti-HBVc, Anti-HIV, T-supressor cell antigen, T-cell antigenratio, T-helper cell antigen, Anti-HCV, Pyrogens, p24 antigen,Muramyl-dipeptide.

Exemplary diabetes markers include without limitation C-Peptide,Hemoglobin Alc, Glycated albumin, Advanced glycosylation end products(AGEs), 1,5-anhydroglucitol, Gastric Inhibitory Polypeptide, Glucose,Hemoglobin, ANGPTL3 and 4.

Exemplary inflammation markers include without limitation Rheumatoidfactor (RF), Antinuclear Antibody (ANA), C-reactive protein (CRP), ClaraCell Protein (Uteroglobin).

Exemplary allergy markers include without limitation Total IgE andSpecific IgE.

Exemplary autism markers include without limitation Ceruloplasmin,Metalothioneine, Zinc, Copper, B6, B12, Glutathione, Alkalinephosphatase, and Activation of apo-alkaline phosphatase.

Exemplary coagulation disorders markers include without limitationb-Thromboglobulin, Platelet factor 4, Von Willebrand factor.

In some embodiments a marker may be therapy specific. COX inhibitorsinclude without limitation TxB2 (Cox-1), 6-keto-PGF-1-alpha (Cox 2),11-Dehydro-TxB-1a (Cox-1).

Other markers of the present include without limitation Leptin, Leptinreceptor, and Procalcitonin, Brain 5100 protein, Substance P,8-Iso-PGF-2a.

Exemplary geriatric markers include without limitation, Neuron-specificenolase, GFAP, and S100B.

Exemplary markers of nutritional status include without limitationPrealbumin, Albumin, Retinol-binding protein (RBP), Transferrin,Acylation-Stimulating Protein (ASP), Adiponectin, Agouti-Related Protein(AgRP), Angiopoietin-like Protein 4 (ANGPTL4, FIAF), C-peptide, AFABP(Adipocyte Fatty Acid Binding Protein, FABP4) Acylation-StimulatingProtein (ASP), EFABP (Epidermal Fatty Acid Binding Protein, FABP5),Glicentin, Glucagon, Glucagon-Like Peptide-1, Glucagon-Like Peptide-2,Ghrelin, Insulin, Leptin, Leptin Receptor, PYY, RELMs, Resistin, amdsTfR (soluble Transferrin Receptor).

Exemplary markers of Lipid metabolism include without limitationApo-lipoproteins (several), Apo-A1, Apo-B, Apo-C-CII, Apo-D, Apo-E.

Exemplary coagulation status markers include without limitation FactorI: Fibrinogen, Factor II: Prothrombin, Factor III: Tissue factor, FactorIV: Calcium, Factor V: Proaccelerin, Factor VI, Factor VII:Proconvertin, Factor VIII:, Anti-hemolytic factor, Factor IX: Christmasfactor, Factor X: Stuart-Prower factor, Factor XI: Plasma thromboplastinantecedent, Factor XII: Hageman factor, Factor XIII: Fibrin-stabilizingfactor, Prekallikrein, High-molecular-weight kininogen, Protein C,Protein S, D-dimer, Tissue plasminogen activator, Plasminogen,a2-Antiplasmin, Plasminogen activator inhibitor 1 (PAI1).

Exemplary monoclonal antibodies include those for EGFR, ErbB2, andIGF1R.

Exemplary tyrosine kinase inhibitors include without limitation Abl,Kit, PDGFR, Src, ErbB2, ErbB 4, EGFR, EphB, VEGFR1-4, PDGFRb, FLt3,FGFR, PKC, Met, Tie2, RAF, and TrkA.

Exemplary Serine/Threoline Kinas Inhibitors include without limitationAKT, Aurora A/B/B, CDK, CDK (pan), CDK1-2, VEGFR2, PDGFRb, CDK4/6,MEK1-2, mTOR, and PKC-beta.

GPCR targets include without limitation Histamine Receptors, SerotoninReceptors, Angiotensin Receptors, Adrenoreceptors, MuscarinicAcetylcholine Receptors, GnRH Receptors, Dopamine Receptors,Prostaglandin Receptors, and ADP Receptors.

In a separate embodiment, a method of monitoring more than onepharmacological parameter useful for assessing efficacy and/or toxicityof a therapeutic agent is provided. For example, a therapeutic agent caninclude any substances that have therapeutic utility and/or potential.Such substances include but are not limited to biological or chemicalcompounds such as simple or complex organic or inorganic molecules,peptides, proteins (e.g. antibodies) or a polynucleotides (e.g.anti-sense). A vast array of compounds can be synthesized, for examplepolymers, such as polypeptides and polynucleotides, and syntheticorganic compounds based on various core structures, and these can alsobe included as therapeutic agents. In addition, various natural sourcescan provide compounds for screening, such as plant or animal extracts,and the like. It should be understood, although not always explicitlystated that the agent is used alone or in combination with anotheragent, having the same or different biological activity as the agentsidentified by the inventive screen. The agents and methods also areintended to be combined with other therapies. For example, smallmolecule drugs are often measured by mass-spectrometry which can beimprecise. ELISA (antibody-based) assays can be much more accurate andprecise.

Physiological parameters according to the present invention includewithout limitation parameters such as temperature, heart rate/pulse,blood pressure, and respiratory rate. Pharmacodynamic parameters includeconcentrations of biomarkers such as proteins, nucleic acids, cells, andcell markers. Biomarkers could be indicative of disease or could be aresult of the action of a drug. Pharmacokinetic (PK) parametersaccording to the present invention include without limitation drug anddrug metabolite concentration. Identifying and quantifying the PKparameters in real time from a sample volume is extremely desirable forproper safety and efficacy of drugs. If the drug and metaboliteconcentrations are outside a desired range and/or unexpected metabolitesare generated due to an unexpected reaction to the drug, immediateaction may be necessary to ensure the safety of the patient. Similarly,if any of the pharmacodynamic (PD) parameters fall outside the desiredrange during a treatment regime, immediate action may have to be takenas well.

Being able to monitor the rate of change of an analyte concentration orPD or PK parameters over a period of time in a single subject, orperforming trend analysis on the concentration, PD, or PK parameters,whether they are concentrations of drugs or their metabolites, can helpprevent potentially dangerous situations. For example, if glucose werethe analyte of interest, the concentration of glucose in a sample at agiven time as well as the rate of change of the glucose concentrationover a given period of time could be highly useful in predicting andavoiding, for example, hypoglycemic events. Such trend analysis haswidespread beneficial implications in drug dosing regimen. When multipledrugs and their metabolites are concerned, the ability to spot a trendand take proactive measures is often desirable.

In some embodiments, the present invention provides a business method ofassisting a clinician in providing an individualized medical treatment.A business method can comprise post prescription monitoring of drugtherapy by monitoring trends in biomarkers over time. The businessmethod can comprise collecting at least one pharmacological parameterfrom an individual receiving a medication, said collecting step iseffected by subjecting a sample of bodily fluid to reactants containedin a fluidic device, which is provided to said individual to yield adetectable signal indicative of said at least one pharmacologicalparameter; and cross referencing with the aid of a computer medicalrecords of said individual with the at least one pharmacologicalparameter of said individual, thereby assisting said clinician inproviding individualized medical treatment.

The devices, systems, and methods herein allow for automaticquantification of a pharmacological parameter of a patient as well asautomatic comparison of the parameter with, for example, the patient'smedical records which may include a history of the monitored parameter,or medical records of another group of subjects. Coupling real-timeanalyte monitoring with an external device which can store data as wellas perform any type of data processing or algorithm, for example,provides a device that can assist with typical patient care which caninclude, for example, comparing current patient data with past patientdata. Therefore, also provided herein is a business method whicheffectively performs at least part of the monitoring of a patient thatis currently performed by medical personnel.

EXAMPLE 1

In this example, a device, method, and system of the invention are usedto perform an assay for human VEGFR2. The example demonstrates a type ofassay that can be performed at the point of care. The capture surface ofan assay unit can be coated onto the assay unit according to the assay,this example a VEGFR2 assay. The inner surface of the assay unit (madefrom injection molded polystyrene similar to example in FIG. 3A) wasexposed to a succession of coating reagents by aspiration and pneumaticejection. Twenty microliters of each coating reagents were drawn intoassay units and incubated at room temperature for 10 minutes. Thecoating reagents used in this example are, as used in succession,Neutravidin (20 ug/mL) in Carbonate-Bicarbonate buffer (pH 9),biotinylated “capture antibody” (a monoclonal antibody directed toVEGFR2 at 20 ug/mL) in Tris buffered saline, (pH 8), and a “fixative”reagent containing 3% bovine serum albumin in Tris-buffered saline.After the succession of coatings, the assay units were dried by exposureto dry air and stored desiccated.

Samples for analysis are then distributed to the assay unit diluted in asolution of 50 mM tris-buffer (pH 8) containing bovine serum albumin andisotonic sucrose for 20 minutes. In a reagent unit comprising aconjugate, a solution of Alkaline phosphatase (bovine intestine)-labeledmonoclonal antibody directed to VEGFR2 (binding to a distinct epitope tothe antibody of the capture surface) at 250 ng/mL in a stabilizerreagent from Biostab is provided to the assay unit for 10 minutes. Afterthe conjugate has been allowed to bind with the complex of the analytebound to the capture surface, the assay unit was washed with a solutioncontained in a reagent unit (commercially available wash buffer fromAssay Designs). The assay unit was washed 5 times. Then the assay unitwas moved to collect and mix with another reagent contained in adifferent reagent, a solution of a commercially available luminogenicsubstrate for alkaline phosphatase (KPL Phosphaglo), and incubated for10 minutes. The reaction of the assay in the assay unit was thendetected by a detector assembly of the invention.

FIG. 12 demonstrates the VEGFR2 assay response using the method of theexample. The x axis scale is VEGFR2 concentration (pg/mL); the y scaleis relative luminescence (counts). The curve was used to calibrate themodular assay unit and reagent units.

EXAMPLE 2

An assay for human P1GF was performed using the assay units and reagentunits of the invention and read in a commercial instrument. In parallel,an assay using the same reagents was done in prototype disposablecartridges (as described below) in a prototype reader. Analyteconcentrations were 0, 4, 80 and 400 pg/mL respectively. Themeasurements illustrated in FIG. 13 were used to calibrate an assay unitand reagent unit necessary for conducting an assay for human P1GF.

EXAMPLE 3

Magnetizable beads are 1.3 um diameter BioMag magnetic particles fromBangs Laboratories. Beads are coated (by the manufacturer) withanti-Rabbit IgG. Beads are dispersed at 14 mg/mL in tris-bufferedsucrose (or, alternatively, tris buffered saline) containing 3% bovineserum albumin and rabbit anti-human red blood cell IgG, from CedarLaneat >=1.15 mg/mL. Aliquots (10 uL of this dispersion were dispensed intoconical tubes and lyophilized (frozen in liquid N2 and lyophilized forapproximately 24 hrs. at −70 C) prior to insertion into a slot in thecartridge housing. The rabbit antibody binds both to the red cells andto the anti-rabbit IgG-coated beads and forms a co-agglutinate of beadsand red cells.

The lyophilized magnetizable bead pellet was re-suspended by adding 20uL of whole blood then aspirating and dispensing at least 8 times(approximately 1.5 min) into a conical tube.

Blood was separated by placing the tip (in a vertical orientation) in astrong, horizontally oriented magnetic field. Typically 8 uL ofessentially red cell free plasma with no observable hemolysis wasrecovered from a 20 ul blood sample (70% yield). Recovery of analytes(compared to plasma not exposed to the magnetic separation) was close to100% for Protein-C, VEGF, P1GF, Insulin, GIP and G1P-1.

EXAMPLE 4

Serial dilution of a sample for analyses of an analyte can be carriedout in a system as described herein. C-reactive protein (CRP) is anacute-phase marker. Normal levels are in the high ng/mL to low ug/mlrange. In any acute disease process, the human liver produces CRP andlevels in blood can increase to hundreds of ug/ml. CRP has presentedissues for prior art POC analytic systems because of the wide dynamicrange of analyte to be measured (>10⁵-fold).

A system as described herein comprising a fluid transfer device and acartridge or device with arrays of assay and reagent units wasdeveloped. Assay tips having monoclonal anti-CRP bound to their innersurface were mounted in cartridge together with a detector-antibodysolution (alkaline-phosphatase labeled monoclonal anti-CRP (having adifferent epitope specificity than that on the tips), a wash solutionand a chemiluminogenic alkaline phosphatase (PhosphaGLO™) substrate fromKPL.

To assay CRP, the cartridges were loaded with pre-diluted solutions ofCRP used without further dilution. The cartridges were processed by asystem. Successively the CRP solution (10 uL), detector antibody (12 uL)were drawn into the tips incubated for 10 min at 34° C. then discarded.The tips were washed by four aspirations of 20 uL wash solution before15 uL of substrate was aspirated into the tips. After 10 min at 37° C.,light emission was measured by the instrument for 5 s. CRP concentrationwas plotted against the assay signal (photon counts) and the data fittedto a 5-term polynomial function as shown below to generate a calibrationfunction as shown in FIG. 14.

EXAMPLE 5

An experiment was then executed using serial dilutions of a samplecontaining highly concentrated analyte to obtain an unambiguous assayresponse in a system and device as described herein. Solutions of CRP(20 uL) were loaded into cartridges and serially diluted by theinstrument (to dilutions of 1:50, 250, 750 and 1500-fold respectively).The diluted solutions were then processed as in Example 4. When thediluted CRP concentration exceeded the calibration range of the assay(300 ng/mL), a downward response was seen (as shown below; data from twoinstruments).

The response as shown in FIG. 15 can be modeled using a modification ofthe Scatchard binding isotherm (S/Smax=C/(C+C0.5). The modificationassumes that the response of the assay is linearly proportional to theconcentration of the detector antibody, as is the case in this example(data not shown). Any carry-over of CRP in the diluted sample into thenext reagent (detector antibody) will react rapidly with the reagentrendering it incapable of binding to antigen bound to the solid phaseantibody. The reduction in effective concentration is reduced inproportion to the CRP carried-over and can be accounted for with afactor (D−C*f)/D.

Therefore, S=Smax*(C/(C+C0.5))*(D−C*f)/D, wherein S is the assay signal,Smax is the maximum signal (corresponding to zero carry-over), C is theconcentration of analyte, C0.5 is the concentration for half-maximalsignal (no carry-over), D is the detector antibody concentration, and fis the fractional carryover.

Values used to fit the data, were derived by optimizing each of the fourparameters below using the technique of minimization of least squaredifferences between the data and the model fit. As can be seen in FIG.15, an excellent fit was achieved and the values of the parameters Smax,C0.5 and D (see table 2) are close to the values that can be estimatedfrom the maximum signal reached, the observed C0.5 and the knowndetector antibody concentration. This model estimated the extent ofcarry-over as 0.034% (decimal 3.83E-04).

TABLE 1 Best fit parameters to model describing biphasic CRP assayresponse Parameter Value Units Smax 7.24E+05 Counts C0.5 5.02E+01 ng/mLD 5.72E+00 ng/mL f 3.83E−04

Data can be then be viewed according to the dilution used to achieve thefinal concentration in each assay tip, and for each dilution level theresponses fit to the same response showing that the dilutions areaccurate and precise as shown in FIG. 16.

The model as described herein can be used to compute responses for anygiven dilution and set up algorithms to ensure that the analyteconcentration in any tip within the calibration range. Graphic means ofrepresenting the data are shown in FIG. 17, wherein the normalized assayresponse (B/Bmax) is plotted against the log normalized concentration(C/C0.5) for relative dilutions: 1:1 (solid line), 5:1 (dashed line),and 25:1 (dotted line). FIGS. 18 and 19 illustrate a similar example asFIG. 17 at different normalized concentrations. Simple patternrecognition algorithms can be used to identify valid data for highconcentration samples. For example, for most of the dose-response, thesignal decreases with dilution. When signal for any dilution equal orexceed that of the next higher dilution, the lower dilution result isrejected. In another example, concentrations derived by using thecalibration function shown in Example 4, should correspond within somesystem imprecision with the known dilutions. If the calculatedconcentration for a low dilution is lower than would correspond withthose for higher dilutions, the lower dilution result can be rejected.

When the assay dose-response approaches a maximum, the slope of theconcentration (AC/AS) versus signal increases. For assays in which therelative variation in signal (AS/S) is essentially constant (for examplesome instances of the system as described) this translates to a biggervariation in the calculated concentration result at higherconcentrations. As provided herein, dilution or serial dilution canprovide a concentration precision as achieved by immunoassays at signallevels significantly greater (for example, >10-fold) higher than theblank (zero analyte) signal but not close to the maximum signal (forexample<0.3*Max. signal). Serial dilution can allow the assay signal tobe in this range.

By making several estimates of the analyte concentration from differentdilutions, an average value can be obtained. An average value can alsobe achieved by making replicate measurements at a single dilution level.In some instances, a serial dilution approach as offered by the methods,systems, and device described herein can often eliminate errors due tonon-linearity of dilution due to (for example) matrix effects from thesample.

EXAMPLE 6

Fluorescein is a well-known chemical and high affinity antibodies areknown which are specific for the molecule. By attaching severalfluorescein moieties to a protein such as albumin, an artificial analyteis created that can be measured by ELISA. The example herein is set upon a microtiter plate to show the feasibility of such an assay and iseasily translatable to a device or system of the invention as describedherein.

Anti-fluorescein monoclonal antibody was attached to wells of 384-wellmicrotiter plates to create a capture surface. An assay is performed byadding a series of solutions to the wells and incubating at roomtemperature for 10 min at each stage when necessary. 30 ul of knownconcentrations of a commercially available preparation offluorescein-labeled bovine albumin (sample) with a ratio of about fivefluoresceins per molecule were added to the wells. After mechanicalremoval of the sample, 30 ul of alkaline phosphatase-labeledanti-fluorescein (detector antibody) was added at a concentration of 100ng/ml. After removal of the detector antibody, the wells were washedthree times 40 ul of wash solution (“Wash Buffer” Cat#80-1351 [AssayDesigns, Ann Arbor, Mich.] diluted 1:20 before use). PhosphaGLO™ (40 uL)substrate was then added and the assay response was then read in an M5spectro-luminometer for 0.5 s. The assay response is shown in FIG. 20.

Fluorescein-labeled albumin (5 uL at various concentrations up to 80ng/mL) dissolved in Tris-buffered saline containing bovine albumin at 3mg/mL (buffer) was placed in polypropylene tubes and dried by exposureto low humidity air overnight. Complete drying was verified by weighingmany tubes before and after drying and verifying the appropriate weightloss and a near-constant final weight was achieved. The analyte wasrecovered by adding 5 uL water, 20 uL human serum and 180 uL buffer andmixing. Control experiments were made by mixing 5 uL aliquots of analytesolution with 20 uL serum and 180 uL buffer.

Analyte recovery was measured using the assay as described herein. Asshown below, the recovery of assay signal (and analyte) is essentiallyquantitative at all concentrations. It can be desirable to have goodrecovery (>90%), which is precise (<2% CV in recovery). In someinstances, the assay dose-response is linear over the range of interestby having a low concentration of analyte and excess of the reagents. Forexample, a linear assay dose-response can be achieved by havingsufficient capacity for antigen binding on the capture surface such thateven at the highest level of analyte only a moderate proportion (forexample, <30%) of sites are occupied at the end of the binding reaction.As described herein, for analytes in the ng/mL range and assays withshort incubation times (<say 30 m) this condition is achieved withcapture surfaces coated as described previously. In another example,sufficient concentration of detector antibody such that theconcentration is not significantly depleted during the detector antibodyincubation (for example, <30% of the reagent binds to the surface at thehighest antigen levels), and this condition can be satisfied by use ofdetector antibody concentrations in about 5 to 100 ng/mL. In yet anotherexample, a linear assay dose-response can be achieved by havingdevelopment of a signal less than the linear response of the detector(for example, a PMT with up to about 4 million photons per second). Asdescribed herein, systems and methods can fall within this range. In yetanother example, a linear assay dose-response can be achieved bydevelopment of a signal sufficiently high as to be precisely measured(for example, photon count rates greater than about 1,000 per second).

Assay tips (as described herein) were coated by aspiration of thefollowing succession of reagents: 20 uL 5 ug/mL Rabbit anti-fluorescein(Molecular Probes # A6413) in carbonate buffer pH 9, 20 uL 3% bovinealbumin in tris-buffered saline pH 8, and 20 uL 2.5 ug/mL bovine albuminlabeled with fluorescein (Sigma-Aldrich A9771), each followed byincubation for 10 m and ejection of liquid. The tips were then washedthree times by aspiration of bovine albumin in tris-buffered saline pH 8followed by incubation 3% bovine albumin in tris-buffered saline pH 8.Tips were then dried as described herein. These tips were used to assaysamples containing goat anti-fluorescein by incubation of 20 uL aliquotsof the following solutions in sequence: goat anti-fluorescein (sample)in tris-buffered saline pH 8 containing 3% BSA, alkaline phosphataselabeled Rabbit-anti-goat fluorescein at 100 ng/mL in Stabilzyme™ (acommercially available solvent), washing four times with Wash Buffer,and PhosphaGLO™ alkaline phosphatase chemiluminogenic substrate, eachwith an incubation at room temperature for 10 min. The assay wasevaluated by measuring photons produced over about 10 s in theinstrument using a photomultiplier tube in Molecular Devices M5luminometer by placing each tip in a custom-modified frame which fitsthe instrument microtiter plate stage and the results are demonstratedin FIG. 21. In this example, FIG. 21 shows a linear response similar tothat in FIG. 20.

TABLE 2 Configurations of assays for candidate control analytes Capturesurface reagent 1 Capture surface reagent 2 Analyte Detector:APase-labeled Anti-fluorescein Fluorescein-labeled albuminAnti-fluorescein Anti-fluorescein Fluorescein-labeled albuminAnti-fluorescein (species X) Anti X-Ig Avidin Biotinylated-species X-IgGAnti X-Ig Anti-biotin Biotin-labeled albumin Anti-biotin or StreptavidinAnti-digoxin Digoxin-labeled albumin Anti-digoxin Fluorescein-labeledalbumin Anti-fluorescein (species X) Anti-X-Ig Anti-biotin Biotinylatedanti-fluorescein Anti-fluorescein (species X) Anti-X-Ig

EXAMPLE 7

This example illustrates the predictability of response from animmunoassay for CRP using assay tips as described herein followinginitial addition of reagents, removal of the reaction product, washingthe tips then reintroduction of some or all assay components. The assaysequence was: tips were incubated in prototype instruments at 34 C for10 min in succession with (1) sample (CRP 0.3, 3, 30, 150 and 300ug/mL), diluted by the instrument 500 then 2000-fold (2) alkalinephosphatase labeled rabbit anti-goat IgG [“Dab”] (5 ng/mL) then washedthree times and (3) with PhosphaGLO™ alkaline phosphatasechemiluminogenic substrate [“Substrate”]. The experiment was performedon several instruments which also read the proton production rate over10 seconds after step 3. Final (in tip) CRP concentrations were 0.15,0.6, 1.5, 6, 15, 60, 75, 300 and 600 ng/mL and glow levels ranged from2,000 to 600,000 counts/0.5 sec. In some experiments, after step (3) inthe assay, the reaction product was discarded and variously steps 3(diamonds and solid line), steps 2+3 (squares and dashed line), or steps1+2+3 (triangles and dotted line) were repeated and ther esults arepresented as re-processed assay signal versus original assay signal asshown in FIG. 22.

The re-processed assay signals were linearly related (proportional) tothe original assay signal. The second substrate addition gave a highersignal relative to the original whereas reprocessed assays in which Daband substrate were both introduced or those where sample, Dab andsubstrate were all reintroduced gave lower signals than the original. Inan example using this method, all steps in an assay sequence can beexamined for quality control to understand if they went as expectedaccording to the expected relationship between the first and subsequentiterations of assay steps.

For example as described herein, if an assay step has not happenedproperly, then the assay result can either be rejected as incorrect orthe later iterations of the assay result can be used as the appropriateassay response.

An immunoassay for C-reactive protein was preformed in a system asdescribed herein. Six equivalent assay tips were incubated in successionwith sample (200 ng/mL CRP), alkaline phosphatase labeled rabbitanti-goat IgG then washed and incubated with PhosphaGLO™ alkalinephosphatase chemiluminogenic substrate. Incubations were for 10 min at34 C. The experiment was performed on three instruments which also readthe proton production rate over 10 seconds On average about 40,000counts (photons) per 0.5 second read time were detected. In thisexample, the glow level on tips one and two on instrument three gaveclearly different results as shown in Table 3. The instrument was thenused to wash the tips and to introduce fresh PhosphaGLO™ substrate(aspiration 2). Results are presented as ratios of glow rate for eachtip to the average for the six tips on each respective instrument. Afterthe second aspiration, tips one and two gave results in line with theother four in instrument three indicating that whatever problem had beenresponsible for low signal in tips one and two had been rectified.

TABLE 3 Recovery of appropriate signal from malfunctioning tips Signal,Ratio to average Instrument # 1 2 3 3 Aspiration # 1 1 1 2 Tip # 1 1.0020.988 0.460 1.043 2 0.848 1.045 0.917 0.929 3 0.959 0.893 1.141 1.035 41.062 1.067 1.103 1.028 5 1.049 0.981 1.171 1.022 6 1.079 1.025 1.2070.942 CV, % 8.6 6.2 28.3 5.0

What is claimed is:
 1. A method of detecting at least two analytespresent at different concentrations in a small-volume biological sample,the method comprising: providing a cartridge comprising a housingcarrying i) a sample collection unit containing ii) a biological samplehaving a volume of no more than about 500 microliters (μL), iii) anarray of individual, addressable reagent units, and iv) an array ofindividual, addressable assay units, wherein each of said addressablereagent units is addressed to correspond to an addressable assay unit,and wherein said reagent units and said assay units provide all thereagents and assay units required for analyzing said biological samplefor the presence of said at least two analytes; positioning saidcartridge in a benchtop instrument effective that a fluid transferdevice in said benchtop instrument may transfer at least a portion ofsaid biological sample from said sample collection unit; transferring atleast a portion of said biological sample from said sample collectionunit to an individual assay unit to provide a first sample portion andat least a second sample portion, wherein said second sample portion isnot in fluid communication with said first sample portion; diluting, byat least about 10-fold, at least one of said first sample portion orsaid second sample portion effective that signals produced by assaysindicative of said at least two analytes are within detectable ranges;performing a first assay for detecting the presence of a first analytein said first portion of the biological sample; performing a secondassay for detecting the presence of a second analyte in said secondportion of the biological sample; detecting a signal indicative of thepresence of said first analyte; detecting a signal indicative of thepresence of said second analyte; whereby the presence of each of said atleast two analytes is detected in said small-volume biological sample.2. The method of claim 1, further comprising providing a protocol fordirecting the fluid transfer device in the performance of assays for thepresence of said at least two analytes.
 3. The method of claim 1,wherein the at least two analytes are present at concentrations thatdiffer by at least 2 orders of magnitude.
 4. The method of claim 1,wherein performing said first or said second assay comprisestransferring a reagent or transferring at least a portion of a sampleusing said fluid transfer device.
 5. The method of claim 1, wherein saidtransferring comprises one or more of moving at least a portion of asample, moving an assay unit, and transferring reagent by an automatedprocess.
 6. The method of claim 5, wherein said fluid transfer devicecomprises an automated pipette, and said transferring comprises anautomated process carried out by a user-defined protocol.
 7. The methodof claim 1, wherein said fluid transfer device comprises a programmableprocessor.
 8. The method of claim 7, wherein said programmable processorcomprises instructions or commands for the operation of said fluidtransfer device.
 9. The method of claim 2, wherein providing a protocolfurther comprises selecting a protocol effective to provide a selectedprotocol for directing the performance of said fluid transfer device.10. The method of claim 9, wherein said cartridge comprises anidentifier, and wherein said protocol is automatically selected bydetecting said identifier and selecting a protocol from a plurality ofprotocols associated with the identifier.
 11. The method of claim 10,wherein said protocol is remotely selected following communication of anidentifier to an external device having a plurality of protocolsassociated with said identifier, and selecting a protocol to be run onthe fluid transfer device from said plurality of protocols.
 12. Themethod of claim 11, wherein said selected protocol is electronicallyreceived by said benchtop instrument.
 13. The method of claim 12,wherein said first assay and said second assay are performed at apoint-of-care (POC) location according to said selected protocol. 14.The method of claim 1, comprising providing said cartridge at apoint-of-care (POC) location.
 15. The method of claim 1, wherein saiddetecting is performed at a point-of-care (POC) location.
 16. The methodof claim 14, wherein said point-of-care (POC) location is a medicaloffice or a laboratory.
 17. The method of claim 15, wherein saidpoint-of-care (POC) location is a medical office or a laboratory. 18.The method of claim 1, wherein said signal indicative of the presence ofsaid first analyte or signal indicative of the presence of said secondanalyte, or both, comprises an optical signal.
 19. The method of claim1, wherein said first assay or said second assay comprises aquantitative assay.
 20. The method of claim 1, wherein performance ofsaid first assay and of said second assay is completed within a periodof time of less than about 1 hour.
 21. The method of claim 1, whereinperformance of said first assay and of said second assay is completedwithin a period of time of less than about 30 minutes, and the at leasttwo analytes are present at concentrations that differ by at least 5orders of magnitude.
 22. The method of claim 1, wherein saidsmall-volume biological sample is a sample of a bodily fluid selectedfrom blood, serum, saliva, urine, gastric and digestive fluid, tears,stool, semen, vaginal fluid, interstitial fluids derived from tumoroustissue, and cerebrospinal fluid.
 23. The method of claim 1, wherein saidsmall-volume biological sample has a volume of between about 1 μL toabout 100 μL.
 24. The method of claim 1, wherein said at least twoanalytes comprise an analyte selected from polypeptides, glycoproteins,polysaccharides, lipids, nucleic acids, biomarkers, genes, proteins,drugs, prodrugs, pharmaceutical agents, drug metabolites, expressedproteins, cell markers, antibodies, hormones, cholesterol and othermetabolites, and combinations thereof
 25. The method of claim 1, whereineach of said at least two analytes comprise an analyte selected frompolypeptides, glycoproteins, polysaccharides, lipids, nucleic acids,biomarkers, genes, proteins, drugs, prodrugs, pharmaceutical agents,drug metabolites, expressed proteins, cell markers, antibodies,hormones, cholesterol and other metabolites, and combinations thereof.26. The method of claim 1, wherein said small-volume biological sampleis a fingerstick blood sample.
 27. The method of claim 1, wherein saidhousing of said cartridge is generic for all assays and is configured tohold a sample collection unit, a plurality of addressable assay units,and a plurality of addressable reagent units, wherein at least one ofsaid assay units and at least one of said reagent units are movablerelative to each other.
 28. The method of claim 1, wherein saiddetecting comprises an assay for the detection of an analyte indicativeof a micro-organism or virus.
 29. The method of claim 1, wherein saiddetecting comprises an assay for the detection of an analyte indicativeof an infectious disease.
 30. The method of claim 1, wherein saiddetecting comprises an assay for the detection of an analyte indicativeof an infectious disease condition.
 31. The method of claim 22, whereinsaid small-volume biological sample is obtained from a mammal.
 32. Themethod of claim 31, wherein said mammal is a human.
 33. The method ofclaim 31, wherein said mammal is a farm animal.
 34. The method of claim31, wherein said mammal is a pet animal.
 35. The method of claim 28,wherein said micro-organism or virus is a pathogen.
 36. The method ofclaim 35, wherein said pathogen is selected from the group of pathogensconsisting of Staphylococcus epidermidis, Escherichia coli,methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcusaureus, Staphylococcus hominis, Enterococcus faecalis, Pseudomonasaeruginosa, Staphylococcus capitis, Staphylococcus warneri, Klebsiellapneumoniae, Haemophilus influenzae, Staphylococcus simulans,Streptococcus pneumoniae, Candida albicans, Stenotrophomonasmaltophilia, Haemophilis parainfluenzae, Serratia marcescens,Haemophilis parahaemolyticus, Enterococcus cloacae, Moraxiellacatarrhalis, Citrobacter freundii, Enterococcus faecium, Klebsellaoxytoca, Pseudomonas fluorscens, Neiseria meningitidis, Streptococcuspyogenes, Pneumocystis carinii, Klebsella pneumoniae Legionellapneumophila, Mycoplasma pneumoniae, and Mycobacterium tuberculosis.